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Mungbean residue effects on the growth parameters of a succeeding mungbean crop Bantilan, Roberto T. 1979

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MUNGBEAN RESIDUE EFFECTS ON THE GROWTH PARAMETERS OF A. SUCCEEDING MUNGBEAN :CROP by ROBERTO T. BANTILAN B.S.A., Mindanao I n s t i t u t e o f T e c h n o l o g y , 1960 C o t a b a t o , P h i l i p p i n e s A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n t h e F a c u l t y o f G r a d u a t e S t u d i e s Department o f PLANT SCIENCE We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA November, 1979 (c) R o b e r t o T. B a n t i l a n In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of The University of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date / 4 TUIM^AJIZAJ / < ? 7 f ABSTRACT I n v e s t i g a t i o n s of mungbean (vigna radiata (L.) Wilczek) r e s i d u e s , which have been found to have adverse e f f e c t s and cause s u b s t a n t i a l y i e l d r e d u c t i o n on subsequent mungbean crops grown i n r a p i d r o t a t i o n , were undertaken to determine the source of phytotoxin and study i t s e f f e c t s on growth parameters. There was no e f f e c t on p l a n t s grown i n pots of s t e a m - s t e r i l i z e d s o i l t hat had re c e i v e d leachates from a c t i v e l y growing mungbean p l a n t s i n sand c u l t u r e , or i n pots that had re c e i v e d leachates of roots and leaves decomposing i n sand. E f f e c t s from residues of previous mungbean crops were demonstrated when succeeding mungbean crops were grown such t h a t t h e i r roots were i n d i r e c t p h y s i c a l contact w i t h the re s i d u e . P l a n t s grown i n s o i l i n which r o o t - l e a f residue mix had been incubated f o r one week p r i o r t o seeding were about 50% of c o n t r o l i n t o t a l dry weight a t any sampling date. T o t a l dry weight was f u r t h e r reduced to about 40% when i n c u b a t i o n time was increased to three weeks. Separate experiments w i t h root and l e a f residues showed t h a t l e a f residues were about 12.3% more t o x i c than root residues on a propo r t i o n a t e residue weight b a s i s . The combination of l e a f and root residues di d not show a d d i t i v e e f f e c t s . i i i I n c o r p o r a t i o n of the residues i n t o the s o i l prevented normal s e e d l i n g development. P l a n t s growing from r e s i d u e - t r e a t e d s o i l had more a s s i m i l a t e s a l l o c a t e d to the leaves during the ve g e t a t i v e stage compared to those from r e s i d u e - f r e e s o i l . During t h i s stage net a s s i m i l a t i o n r a t e , r e l a t i v e growth r a t e , r e l a t i v e l e a f area growth r a t e , and l e a f area r a t i o became considerably greater than f o r c o n t r o l s . Although, r e l a t i v e l e a f area growth r a t e was inc r e a s e d , which may have been due to more a s s i m i l a t e s being a l l o c a t e d to the le a v e s , the greater magnitude of the increase i n r e l a t i v e growth r a t e over t h a t of the r e l a t i v e l e a f area growth r a t e may account f o r the increase i n the value of net a s s i m i l a t i o n r a t e . This would be p o s s i b l e i f there was a redu c t i o n i n r e s p i r a t o r y l o s s e s , caused by the r e l e a s e of a r e s p i r a t o r y i n h i b i t o r from the r e s i d u e s . TABLE OF CONTENTS i v Page ABSTRACT 1 1 TABLE OF CONTENTS * v LIST OF TABLES v i LIST OF FIGURES v i i ACKNOWLEDGEMENTS i x LIST OF ABBREVIATIONS X INTRODUCTION 1 LITERATURE REVIEW 6 A l l e l o p a t h y 6 Growth A n a l y s i s 16 MATERIALS AND METHODS 21 General Management 21 Experiment l.Root l e a c h a t e t r a n s f e r 23 N u t r i e n t s o l u t i o n 25 Donor pots 26 R e c i p i e n t pots 26 Experimental d e s i g n 27 Experiment 2. Leachate t r a n s f e r from decomposing r o o t and l e a f r e s i d u e s 27 Experiment 3a. S e q u e n t i a l mungbean cr o p p i n g w i t h w i t h r o o t and l e a f r e s i d u e s i n c o r p o r a t e d i n t o the s o i l 28 E s t a b l i s h m e n t of f i r s t crop 28 Second crop 29 Experimental design 29 V Page Experiment 3b. Separate e f f e c t s of le a f and root residues from previous mungbean crop 30 Col l e c t i o n of data 31 RESULTS 34 Leachate transfer 34 Sequential cropping 38 Effe c t s of leaf and root residues 56 DISCUSSION 68 Residue e f f e c t s 68 Growth parameters 71 SUMMARY 79 REFERENCES 81 APPENDIX 86 v i LIST OF TABLES Table Page 1. Top weight (g/pot of 4 p l a n t s ) o f mungbeans grown i n s o i l t hat had received leachates from mungbeans growing on sand c u l t u r e 35 2. Leaf area (dm /pot of 4 pl a n t s ) of mungbeans grown i n s o i l t hat had received leachates from mungbeans growing on sand c u l t u r e . . . . 35 3. Top weight (g/pot of 4 pl a n t s ) of mungbeans grown i n s o i l t h a t had received leachates o f decomposing roots and leaves 36 2 4. Leaf area (dm /pot of 4 plan t s ) of mungbeans grown i n s o i l t hat had r e c e i v e d leachates from decomposing roots and leaves 37 5. Average p l a n t height (cm) o f mungbeans grown i n s o i l t h a t had re c e i v e d leachates from decomposing roots and leaves 37 6. Summary of c o n t r a s t between e f f e c t s of the treatments: w i t h vs without r e s i d u e , and the e f f e c t s of the treatments one- vs three-week i n c u b a t i o n p e r i o d 45 7. Comparison of means of a l l harvests f o r 1-week and 3-week i n c u b a t i o n periods w i t h l e a f and root residues 48 8. Summary of c o n t r a s t between treatments: w i t h vs without l e a f residue and treatments: w i t h vs without root residue 59 9. Dry weight r e d u c t i o n of mungbean a t f i n a l h a r v e s t , i n percent of c o n t r o l , caused by the i n c o r p o r a t i o n i n the s o i l o f l e a f , r o o t and r o o t plus l e a f residues of previous mungbean crop 69 v i i LIST OF FIGURES Figure Page 1. General arrangement and close-up view of the root leachate transfer experiment 24 2. Reduced s i z e of mungbean seedlings at 7DAE grown i n cropped s o i l with root and leaf residues incorporated and incubated for one week 39 3. Reduced cotyledonary leaf expansion of mungbean grown i n the residue-treated s o i l . 14 DAE 39 4. Stunted growth of mungbean grown i n cropped s o i l with root and leaf residues incorporated and incubated for one week. 14 DAE . 40 5. The same treatment as i n Figure 4 at 28 DAE. . . 40 6. Root development of mungbean shown i n Figure 4. . . 41 7. Root development of mungbean at 28 DAE of the same treatment as i n Figure 4 41 8. The e f f e c t of root and leaf residues, incorporated and incubated for three weeks into the s o i l , on the development of mungbean seedlings . . . . 42 9. The same treatment as i n Figure 8 at 7 DAE . . . 42 10. Normal seedling development of mungbean at 7 DAE . . . 43 11. Growth of mungbeans i n cropped s o i l with leaf and root residues of the previous crop incorporated and s o i l that was l a i d fallow, without residues 46 12. Comparison of growth parameters of mungbean grown on s o i l s with residues vs s o i l s without residues 50 13. Total dry weight, leaf area, r e l a t i v e growth rate, r e l a t i v e leaf area growth rate, net assimilation rate, leaf area r a t i o , and a of the succeeding mungbean crop grown on s o i l containing leaf and root residues a f t e r a one-week incubation . . . 52 v i i i F igure Page 14. T o t a l dry weight, l e a f area, r e l a t i v e growth r a t e , net a s s i m i l a t i o n r a t e , r e l a t i v e l e a f area growth r a t e , l e a f area r a t i o , and a o f the succeeding mungbean crop grown on s o i l c o n t a i n i n g l e a f and ro o t residues a f t e r a three-week i n c u b a t i o n p e r i o d 54 15. The e f f e c t of l e a f residue on the t o t a l dry weight, l e a f area, l e a f , stem, and root weights, l e a f weight r a t i o , s p e c i f i c l e a f area r a t i o and l e a f area r a t i o 60 16. The e f f e c t of ro o t residue on the t o t a l dry weight, l e a f area, l e a f , stem and root weights, l e a f weight r a t i o , s p e c i f i c l e a f area r a t i o and l e a f area r a t i o 61 17. Richard's ( f a c t o r ) diagram of root x l e a f i n t e r a c t i o n 64 18. The e f f e c t of l e a f and ro o t residues on r e l a t i v e growth r a t e and net a s s i m i l a t i o n r a t e of the second crop of mungbean 66 19. The e f f e c t of l e a f and root residues on the r e l a t i v e l e a f area growth r a t e and R/Rj-r a t i o of the second crop of mungbean 67 20. Reduction of dry weight o f tops of mungbean as i n f l u e n c e by date of p l a n t i n g 72 21. Component dry weights as percent o f t o t a l dry weight of succeeding mungbean crop as a f f e c t e d by the residue and length o f in c u b a t i o n of previous mungbean crop 75 22. Component dry weights as percent o f t o t a l dry weight of succeeding mungbean crop as a f f e c t e d by the l e a f and/or r o o t residues of the previous mungbean crop 76 ACKNOWLEDGEMENT I would l i k e to thank Dr. V.C. Runeckles, Chairman, Department of Plant Science, The University of B r i t i s h Columbia, under whose supervision this research was carried out, for his patient guidance, c r i t i c i s m , and assistance i n the preparation of this t h e s i s . I wish to thank the members of my graduate committee for reviewing t h i s t h e s i s . I am gratef u l to the International Development Research Centre, Ottawa, Canada, for a scholarship and f i n a n c i a l support for conducting my research. The assistance of Dr. G.W. Eaton i n the s t a t i s t i c a l analysis of my research data i s also hereby acknowledged with gratitude. To my children, Teresa, Roberto, Elena, Marilou, Marietta and Christine (who was only 19 months o l d when I came to UBC), I deeply appreciate t h e i r calm endurance of the long absence of t h e i r father. F i n a l l y , I wish to express my h e a r t f e l t gratitude and deepest appreciation to my wife Cresencia for her moral support and perseverance i n carrying out alone our family r e s p o n s i b i l i t i e s i n my absence. I dedicate t h i s manuscript to Cresencia. LIST OF ABBREVIATION cm Sentimeter DAE days a f t e r emergence dm decimeter g gram ID i n s i d e diameter L l e a f area of a l l leaves LAR l e a f area r a t i o LWR l e a f weight r a t i o mg m i l l i g r a m mm m i l l i m e t e r NAR: E net a s s i m i l a t i o n r a t e NPK n i t r o g e n phosphorous potassium ( f e r t i l i z e r ) OD o u t s i d e diameter PVC p o l y v i n y l c h l o r i d e RGR: R r e l a t i v e growth r a t e R_ r e l a t i v e l e a f area growth r a t e L i SLA s p e c i f i c l e a f area W whole p l a n t weight (dried) W_ dry l e a f weight Wp' dry pod weight WP wettable powder W dry root weight Wc dry stent weight 1 INTRODUCTION M u l t i p l e c r o p p i n g , as p r a c t i c e d by t r a d i t i o n a l s m a l l farmers i n the t r o p i c s , allows crop i n t e n s i f i c a t i o n through maximum u t i l i z a t i o n o f space and time (IRRI, 1973; Sanchez, 1976). The i n h e r e n t problems i n t h i s p r a c t i c e have determined the e v o l u t i o n o f the observed cropping p a t t e r n s among s m a l l farmers i n the t r o p i c a l regions o f the world. F i e l d experiments undertaken by the M u l t i p l e Cropping Department of the I n t e r n a t i o n a l Rice Research I n s t i t u t e (IRRI) at Los Banos, P h i l i p p i n e s have shown the harmful e f f e c t s of c e r t a i n legumes (IRRI, 1973; S i k u r a j a p a t h y , 1974) and d r y l a n d r i c e (Ventura and Watanabe, 1978) on the succeeding crop. The g r e a t e s t e f f e c t was demonstrated t o be on subsequent p l a n t i n g o f the same crop; t h a t i s , i n mungbean-mungbean, cowpea-cowpea o r r i c e - r i c e (dryland) sequences. Sweet pot a t o has been shown t o be a d v e r s e l y a f f e c t e d by pr e v i o u s mungbean o r cowpea crops and cowpea has been shown t o be a d v e r s e l y a f f e c t e d by a p r e v i o u s mungbean crop. The t o l e r a n c e o f mungbean to drought makes i t a d e s i r a b l e crop i n a r i c e - b a s e d cropping p a t t e r n where the farmer i s dependent on a v a i l a b l e r a i n f a l l . The s h o r t growth d u r a t i o n o f some o f the newly developed v a r i e t i e s o f mungbean allows two s u c c e s s i v e crops a f t e r r i c e , where 2 the available s o i l moisture i s not s u f f i c i e n t for a second crop of r i c e . An understanding of the mechanism of the residual e f f e c t of mungbean on a subsequent mung-bean crop w i l l thus be of great benefit to small farmers since only with t h i s understanding w i l l i t be possible to develop ways to eliminate or at le a s t circumvent the problem. Evaluation of the e a r l i e r experiments on residue ef f e c t s conducted at IRRI and at the University of the Philippines tended to provide evidence supporting an a l l e l o p a t h i c mechanism (Runeckles, 1974) although the role of nematodes and s o i l fungi was not conclusively ruled out (Runeckles, 1975). However, the experiments of Ventura and Watanabe (1978) showed that i n mungbeans the i n h i b i t o r y e f f e c t s appeared to be d i r e c t l y dependent on microorganisms i n the s o i l , although i t was not determined whether the microorganisms caused d i r e c t damage to the root system as soil-borne pathogens or caused the pro-duction of toxic substances i n h i b i t o r y to mungbean growth. The phenomenon of the harmful effects of some crop residues i s well-known to farmers. Farmers involved i n cooperative experiments with IRRI i n the study of rice-based cropping patterns are reluctant to include mungbean as a rotation crop (IRRI, 1975) e s p e c i a l l y when the f i e l d i s intended for a tomato crop later; i n the season. 3 In the experimental f i e l d s a t IRRI where t h i s phenomenon has a l s o been observed, the leaves and r o o t s are l e f t on and i n the s o i l a t h a r v e s t ; the stems and pods are removed. The leaves are n a t u r a l l y shed as the pods mature, and accumulate and decompose on the s u r f a c e o f the s o i l . I t i s probable then t h a t only r o o t s and leaves are a s s o c i a t e d w i t h the r e s i d u e problem. However, the p o s s i b i l i t y a l s o e x i s t s t h a t m a t e r i a l s are r e l e a s e d i n t o the s o i l d u r i n g the a c t i v e growth o f the crop and t h a t they p e r s i s t l o n g enough duri n g the growth o f the subsequent crop to induce harmful e f f e c t s . Hence, the f o l l o w i n g p o s s i b l e mechanisms needed to be i n v e s t i g a t e d : 1) the r o l e o f exudates from a c t i v e l y growing r o o t s , 2) the r o l e o f decomposing r o o t r e s i d u e s , 3) the r o l e of decomposing l e a f r e s i d u e s , 4) the i n t e r a c t i v e r o l e s o f both r o o t and l e a f r e s i d u e s . With these p o s s i b l e sources o f p h y t o t o x i n , i t was deemed e s s e n t i a l to study f i r s t the e f f e c t s o f a mungbean crop on the growth parameters o f a succeeding mungbean crop grown under v a r i o u s c o n d i t i o n s o f p o t e n t i a l t r a n s f e r and source o f p h y t o t o x i c a n t s , r a t h e r than to attempt to i d e n t i f y the chemical compounds r e s p o n s i b l e . Hence a s e r i e s of experiments was undertaken i n order to e s t a b l i s h t h a t the phenomenon observed i n the f i e l d c o u l d be reproduced under experimental greenhouse c o n d i t i o n s , and subsequently to determine the r e l a t i v e r o l e s o f r o o t 4 or leaf residues i n inducing the e f f e c t . Since previous f i e l d studies at IRRI had been e s s e n t i a l l y confined to the v i s u a l observations of symptoms of impaired growth and measurement of y i e l d , i t was a n t i c i -pated that the analysis of growth throughout the growing period might reveal how the dynamics of growth were affected, whether or not severe symptoms or y i e l d effects were observed. An additional factor which required investigation related to whether the e f f e c t involved the transport of soluble phytotoxicants (e.g. see Rice, 1974) or was dependent upon physical contact between residues and crop roots as has been found to be the case i n some systems (e.g. see Patrick et a l . , 1963). To achieve these various objectives, the following three experimental approaches were used: 1) The hypothesis that the growing mungbean plant produces phytotoxic root exudates was studied by transfer-ing the leachates from the roots of plants i n donor pots into receptor pots. 2) The hypothesis that only the decomposing materials, i . e . , roots and leaves, produce toxic compounds that are water-soluble and transferable was studied by transfering the leachates from decomposing roots and leaves from donor to receptor pots. 3) The hypothesis that the residues have to be i n contact with the roots of a succeeding crop was studied by i n c o r p o r a t i n g the d r i e d and ground r o o t s and/or l e d i r e c t l y i n t o the pots o f the second crop. 6 LITERATURE REVIEW Allelopathy The term allelopathy refers to the detrimental d i r e c t or i n d i r e c t biochemical effects of one plant (the donor) on the germination, growth, or development of another (receptor) plant (Putnam and Duke, 1978; Rice, 1974). The phenomenon, i t should be pointed out, i s not limited to the interactions between species. While the l i t e r a t u r e i s replete with cases of biochemical interactions between species, several plants are also reported to exhibit autotoxicity as i n the case for example of hedge bindweed (Convolvulus sepium) (Quinn, 1974) , some v a r i e t i e s of r i c e (Sadhu and Das, 1971; Ventura and Watanabe, 1978), and a l f a l f a and timothy (Nielsen et a l . , 1960) to mention a few. The influence of allelopathy i n agriculture was recognized as early as the 5th century BC (Putnam and Duke, 1978). But i t i s only the l a s t twenty years that i t s s i g nificance has caught the increasing attention of plant s c i e n t i s t s and has led to an increasing number of publications and reviews, and to the tr e a t i s e by Rice (1974). Allelopathy has been recognized i n diverse plant habitats, for example, i n deciduous forest (Lodhi, 1976) , i n old f i e l d succession (Rice, 1972; Wilson and Rice, 1968) and i n the a r i d C a l i f o r n i a chapparal (Muller et a l . , 1968). Because of the immensity of the subject of allelopathy i n general, I l i m i t the emphasis of this review to the problems that arise from crop residues. 7 In a griculture, there are numerous reports of the e f f e c t s of crop residues. For example, Nielsen et a l . (1960) reported the e f f e c t s of a l f a l f a , corn, oats, potatoes and timothy on the germination and growth of si x plant species. Patrick and Koch (1958) studied the effects of timothy, corn, rye, and tobacco residues i n s o i l on the r e s p i r a t i o n of tobacco seedlings. McCalla and Army (1961) reported extensively on the e f f e c t of stubble-mulch farming. In the study by Patrick and Koch (1958) , i t was found that the substances capable of i n h i b i t i n g the germination, growth and r e s p i r a t i o n of tobacco seedlings arise under some conditions of decomposition. Species and stage of maturity of plant material, water content and pH of the s o i l , and length of decomposition period were among the important factors which influenced the production of phytotoxic products. Aqueous extracts of macerated undecomposed plant materials were not found to be t o x i c i n t h e i r study. However, the germination and growth experiments of Nielsen et a l . (1960) , using the standard germination technique i n sand, indicated that aqueous extracts of crop residues of a l f a l f a , timothy, oats, corn and potato contained substances toxic to at least one of these crops and soybean. Compared to plants watered with deionized water, extracts from a l f a l f a residue caused the greatest reduction i n shoot and root length and i n percentage germination. They also 8 caused the greatest delay i n germination. Timothy extract was not as harmful as that from a l f a l f a . Oats, corn and potato extracts were s t i l l less harmful, with potato extract the l e a s t . The crop species i n t h i s experiment also showed marked differences i n t h e i r tolerance to the phytotoxic ef f e c t s of the extracts. The order of decreasing resistance to the phytotoxic e f f e c t s i n general was as follows: a l f a l f a , corn, soybeans, peas, oats and timothy. However, a l f a l f a , timothy, corn and oats residues were shown to cause deleterious autotoxic e f f e c t s . As indicated by the ra t i o s of observations made on plants grown with and without extracts, timothy showed the greatest autotoxicity i n terms of rate and percentage of germination, root and shoot length. A l f a l f a extract caused the greatest e f f e c t on root growth but i t s e f f e c t on shoot growth and percent germination was less than that of timothy. Corn and oats showed much less autotoxicity. The examples of the e f f e c t s of crop residues raises the question about the ef f e c t s of stubble-mulch farming. Extensive studies by McCalla and associates (McCalla and Army, 1961) have found that while the practice has been demonstrated to be of p r a c t i c a l value i n reducing s o i l erosion by wind and water, plant residues contain substances,and microorganisms i n the decomposing stubble produce substances that may a f f e c t germination and growth. Guenzy and McCalla (1966) i d e n t i f i e d several phenolic compounds from stubble-mulch f i e l d s , and other workers 9 have a l s o r e p o r t e d a number o f p h e n o l i c a c i d s and other r e l a t e d compounds which were shown to be p h y t o t o x i c to v a r i o u s crops from s o i l s c o n t a i n i n g decomposing p l a n t r e s i d u e s (e.g., Borner, 1960; Toussoun e t a l . , 1968; Wang e t a l . , 1967; Whitehead, 1963, 1964; Chou and P a t r i c k , 1976; Chou and L i n , 1976). The r o l e o f microorganisms i n the r e s i d u e problem i s i l l u s t r a t e d by the work o f Cochran e t a l . (1977) w i t h the crop r e s i d u e s common i n e a s t e r n Washington. The study was undertaken t o i n v e s t i g a t e the problem of reduced stand and p l a n t v i g o r i n the n o - t i l l a g e o r reduced t i l l a g e system o f wheat p r o d u c t i o n i n the area. Mats (5-8 cmm t h i c k ) o f crop r e s i d u e o f l e n t i l s , pea, wheat, b a r l e y and bluegrass.- were spread over bare f i e l d o f Palouse s i l t loam s o i l . Water e x t r a c t s o f re s i d u e s and s o i l beneath them were bio a s s a y e d weekly f o r wheat-seedling p h y t o t o x i c i t y and the r e s i d u e s were p l a t e d biweekly t o determine the numbers o f f u n g i , b a c t e r i a , and pseudomonads f o r the succeeding nine months, s t a r t i n g i n September, 1975. A l l r e s i d u e s were found t o produce wheat-seedling r o o t i n h i b i t o r s , but only a f t e r c o n d i t i o n s became f a v o r a b l e f o r m i c r o b i a l growth. The r e s i d u e s were t e s t e d f o r the presence o f p a t u l i n by t h i n l a y e r chromatography but were found to be n e g a t i v e . No f u r t h e r attempt was made t o i d e n t i f y the p h y t o t o x i n i n t h i s experiment. P a t u l i n i s a substance produced by Peniciilium urticae B a i n e r found to be common i n stubble-mulch t i l l a g e i n Nebraska (Norstadt and McCalla, 196 8) and has been suggested to be the cause of as much as 50% i n h i b i t i o n of shoot growth i n wheat. Generally, the a l l e l o p a t h i c compounds i s o l a t e d from plant materials and from s o i l belong to the group known as secondary plant compounds. These include simple phenolic acids, coumarins, terpenoids, f l a -vonoids, al k a l o i d s , cyanogenic glycosides and glu-sosinolates (Rice, 1974; Harborne, 1972). Rice (1974) proposed that the probable biosynthetic pathway of synthesis of the d i f f e r e n t classes of a l l e l o p a t h i c chemicals arise through the acetate or shikimic acid pathways. A l l e l o p a t h i c compounds are not unique chemically. These compounds have also been reported to be involved i n several protective or defensive functions for the plant (Swain, 1977; Rice, 1974). For example, polysaccharides acylated with f e r u l i c acid are suspected to be involved i n the defensive functions of the plant c e l l wall against invading microorganisms (Wood and Granite, 1976) and as phytoalexins (Deveral, 1972; Swain, 1977), while f e r u l i c acid has been i d e n t i f i e d as one of the a l l e l o -pathic agents of decaying l i t t e r of dominant trees i n a lowland forest community (Lodhi, 1978), i n corn and rye residue decomposition (Chou and Patrick, 1976) and i n the phytotoxic e f f e c t s of decomposing r i c e residues i n s o i l (Chou and L i n , 1976). A c c o r d i n g to Putnam and Duke (1978) no one has proven t h a t a l l e l o p a t h i c chemicals are s p e c i f i c a l l y s y n t h e s i z e d as a r e s u l t o f an e x t e r n a l s t i m u l u s . Whether the chemicals i n v o l v e d are end products o f metabolism or are a c t u a l l y s y n t h e s i z e d by the p l a n t f o r a s p e c i f i c f u n c t i o n i s a major unanswered q u e s t i o n i n p l a n t b i o -chemical i n t e r a c t i o n s . I t i s known t h a t a l l e l o p a t h i c chemicals are p o t e n t i a l l y a u t o t o x i c and are shunted i n t o vacuoles to prevent a u t o t o x i c i t y (Whittaker, 1970). A l l e l o p a t h i c e f f e c t s are brought about i n s e v e r a l ways (R i c e , 1974; Putnam and Duke, 1978): by exudation o f v o l a t i l e compounds from l i v i n g p l a n t p a r t s ; by l e a c h i n g o f w a t e r - s o l u b l e t o x i n s from above-ground p a r t s through a c t i o n o f r a i n , f o g o r dew; by exudation o f w a t e r - s o l u b l e t o x i n s from below-ground p a r t s ; o r by r e l e a s e o f t o x i n s through l e a c h i n g from l i t t e r o r as m i c r o b i a l - b y - p r o d u c t s r e s u l t i n g from l i t t e r decomposition. Phyt o t o x i n s once r e l e a s e d must accumulate i n s u f f i c i e n t q u a n t i t y to a f f e c t other p l a n t s , must p e r s i s t f o r some p e r i o d of time, or must be c o n s t a n t l y r e l e a s e d i n order to have l a s t i n g e f f e c t (Rice, 1974). Diver s e techniques have been employed to i d e n t i f y a l l e l o p a t h i c chemicals which cause i n h i b i t o r y e f f e c t s on seed germination, p l a n t growth and development. Rice (1974) has i n c l u d e d d e t a i l e d d e s c r i p t i o n s of many o f the methods employed i n the cases of a l l e l o p a t h y d i s c u s s e d i n h i s t r e a t i s e . Putnam and Duke (1978) have r e c e n t l y presented a comprehensive summary o f the methodology of a l l e l o p a t h y s t u d i e s i n t h e i r review o f a l l e l o p a t h y i n agroecosysterns. The most common method i s t h a t o f cold-water e x t r a c t i o n through simple s o a k i n g , f o r v a r y i n g lengths of time, o f e i t h e r d r i e d o r l i v e p l a n t p a r t s . The e x t r a c t s are then u s u a l l y f i l t e r e d or c e n t r i f u g e d b e f o r e b i o a s s a y i n g i n p e t r i d i s h o r i n f l a t s o f s o i l o r sand or i n n u t r i e n t s o l u t i o n . There are numerous r e p o r t s of the e f f e c t s o f e x t r a c t e d compounds on germination, growth of r o o t s or shoots, and other symptoms (Putnam and Duke, 1978). A v a r i a t i o n o f the cold-water e x t r a c t i o n method i s t h a t o f macerated p l a n t p a r t s p l a c e d i n dishes c o n t a i n i n g moistened c e l l u l o s e sponge t h a t supports a f i l t e r paper on which i n d i c a t o r seeds are sown. Thus cold-water e x t r a c t i o n and b i o -assay are c a r r i e d out s i m u l t a n e o u s l y . Many authors imply t h a t cold-water e x t r a c t i o n s i m u l a t e s the n a t u r a l r e l e a s e of compounds by the a c t i o n of r a i n on f a l l e n p l a n t m a t e r i a l . However, Anderson and Loucks (1966) have demonstrated t h a t e x t r a c t s c o n t a i n i n g unknown and p o s s i b l y high osmotic c o n c e n t r a t i o n s o f non t o x i c compounds, such as s u c r o s e , r e s u l t i n depressions of germination and e a r l y s e e d l i n g development. In a d d i t i o n , at pH values between 5 and 6, sucrose s o l u t i o n (25 m i l l i o s m o l a r ) has been demonstrated to reduce r a d i c l e growth o f l e t t u c e by as much as 75% (Chou and Young, 1974). Most o f the s t u d i e s i n 13 a l l e l o p a t h y r e p o r t e d i n the l i t e r a t u r e do not take i n t o c o n s i d e r a t i o n the r o l e s o f osmotic c o n c e n t r a t i o n and pH of e x t r a c t s used i n the b i o a s s a y s . B i o a s s a y i n g by means of p e t r i d i s h o r n u t r i e n t s o l u t i o n techniques o f m a t e r i a l s e x t r a c t e d by b o i l i n g water (Jackson and Willemsen, 1976) , a u t o c l a v i n g (Kommedahl and Ohman, 1960), o r o r g a n i c s o l v e n t s (Friedman and Horowitz, 19 71) are o t h e r methods which have been used. Hot water and a u t o c l a v i n g e l i m i n a t e m i c r o b i a l decay w h i l e a l l o w i n g i n c r e a s e d d i f f u s i o n o f s o l u b l e compounds i n t o the acqueous phase. The use of o r g a n i c s o l v e n t s - i n the e x t r a c t i o n process permits a l a r g e r number o f compounds to be i s o l a t e d which may be p h y t o t o x i c . But, i n a l l these methods o f e x t r a c t i o n , the p h y t o t o x i c m a t e r i a l s i s o l a t e d may i n c l u d e substances t h a t are not n e c e s s a r i l y the cause of problems under n a t u r a l c o n d i t i o n s (Putnam and Duke, 1978) . In d e t e c t i n g the presence of i n h i b i t o r y substances from below-ground p a r t s , v a r i o u s techniques have been employed. Each method used attempts to prevent water and n u t r i e n t s from b e i n g a l i m i t i n g f a c t o r i n the growth of t e s t p l a n t s so t h a t the observed e f f e c t s can c l e a r l y be a t t r i b u t a b l e t o chemical t o x i c i t y . The most common method i s e x e m p l i f i e d by the s t a i r s t e p system used by B e l l and Koeppe (19 72) i n s t u d y i n g the non-competitive e f f e c t s o f g i a n t f o x t a i l ( S e t a r i a faberii Herm.) on the growth of corn. The system i n v o l v e s the growing o f donor and r e c i p i e n t p l a n t s i n sand i n pots arranged a l t e r n a t e l y i n 14 s t a i r s t e p s so t h a t the n u t r i e n t s o l u t i o n flows from donor to r e c i p i e n t and back to a r e s e r v o i r . The flow o f n u t r i e n t s o l u t i o n i s s e t to a number o f c y c l e s per day. Since the p h y s i c a l aspects o f competition are e l i m i n a t e d i n t h i s system, p o s s i b l e a l l e l o p a t h i c e f f e c t s r e s u l t i n g from the exudation and l e a c h i n g o f p h y t o t o x i n s from one s p e c i e s can be s t u d i e d d i r e c t l y . Some o f the o t h e r methods used i n d e t e c t i n g i n h i b i t o r y substances from r o o t exudates are: (a) growing donor p l a n t s f o r s p e c i f i c times, l e a c h i n g the sand, and e v a l u a t i n g the l e a c h a t e s on r e c i p i e n t p l a n t s i n p e t r i d i s h e s , o r i n s o i l o r sand (Fay and Duke, 1977), (b) growing both donor and r e c i p i e n t p l a n t s i n sand and e v a l u a t i n g the e f f e c t s b e f o r e competition f o r o t h e r growth f a c t o r s occurs (Putnam and Duke, 1974). A s t r a i g h t f o r w a r d method has been used i n examining the r e l e a s e o f o r g a n i c i n h i b i t o r y substances from decom-posing p l a n t m a t e r i a l s . E i t h e r d r i e d or l i v e p l a n t m a t e r i a l s are p l a c e d i n or on s o i l f o r s e l e c t e d time p e r i o d s b e f o r e b i o a s s a y i n g w i t h r e c i p i e n t p l a n t s (Wilson and R i c e , 1968; R i c e , 1972). Although c e r t a i n s t u d i e s have i d e n t i f i e d s p e c i f i c f u n g i i n v o l v e d i n the decomposition o f p l a n t m a t e r i a l s and have e v a l u a t e d by-products of f u n g a l metabolism on p l a n t growth (Norstadt and M c C a l l a , 1962; 1968) there i s d i f f i c u l t y i n determining whether the t o x i c e f f e c t comes from the p l a n t , the microorganism, o r i s the r e s u l t of an a d d i t i v e or s y n e r g i s t i c e f f e c t of both (Rice> 1974). 15 The methodology f o r the study of a l l e l o p a t h y has thus been v a r i e d . There i s s t i l l a need to develop s p e c i f i c and r e l i a b l e procedures f o r the i s o l a t i o n of suspected compounds, and bio a s s a y techniques t h a t are e f f e c t i v e and prove the e x i s t e n c e of t o x i c components more d e f i n i t i v e l y than the e x t r a c t i o n methods used to simulate e f f e c t s observed i n nature. 16 Growth A n a l y s i s Numerous e x c e l l e n t reviews have been w r i t t e n on t h i s s u b j e c t and d i s c u s s i o n can be found i n Evans (1972) , Radford (1967) , Richards (1969) and Sestak e t a l . (1971). Growth a n a l y s i s can be d e s c r i b e d by a s e t o f equa-t i o n s (Evans, 1972) r e p r e s e n t i n g the performance o f the p l a n t ' s f u n c t i o n a l p a r t s ; t h us: RGR = NAR X LAR (dW/dt)(1/W) = (dW/dt)(1/L) x L/W LAR can be f u r t h e r d i v i d e d i n t o i t s components: LAR = SLA x LWR = L/WT x WT/W L L i where RGR = R e l a t i v e Growth Rate; R NAR = Net A s s i m i l a t i o n Rate ( a l s o c a l l e d ULR, U n i t Leaf Rate); E LAR = Leaf Area R a t i o W = Whole p l a n t dry weight L = Leaf area o f a l l leaves SLA = S p e c i f i c Leaf Area LWR = Leaf Weight Ratio WT = Leaf dry weight .U R e l a t i v e growth r a t e i s d e f i n e d as the r a t e o f i n c r e a s e i n dry matter content o f the p l a n t w i t h r e s p e c t to the amount o f dry matter alr e a d y p r e s e n t . I t s use permits the comparison o f the growth of p l a n t s o f d i f f e r e n t s i z e s , s i n c e RGR i s an o v e r a l l measure o f p l a n t performance. 17 The r e l a t i v e growth o f a s s i m i l a t o r y apparatus (and other p l a n t p a r t s ) i s d e f i n e d s i m i l a r l y to the r e l a t i v e growth r a t e o f dry matter accumulation (Sestak e t a l . , 1971). Whitehead and Myerscough (1962) r e p o r t e d t h a t a , the r a t i o o f r e l a t i v e growth r a t e (dry matter b a s i s ) t o r e l a t i v e r a t e of l e a f area i n c r e a s e ( a = R/RT, where RT i s the r e l a t i v e l e a f area growth r a t e ) , i s a parameter of c o n s i d e r a b l e importance. The value of a i n d i c a t e s the amount of dry weight increment t h a t i s i n excess o f the amount r e q u i r e d to maintain the morphogenetic p r o p o r t i o n s of the p l a n t as an e f f i c i e n t p h o t o s y n t h e t i c u n i t . When a = 1 a l l of the dry weight accumulated i s used up i n m a i n t a i n i n g the o v e r a l l m o r p h o l o g i c a l form o f the p l a n t . The l a t e r stages of morphogenesis which e n t a i l the p r o d u c t i o n o f storage organs, and r e p r o d u c t i v e s t r u c t u r e s thus u s u a l l y r e q u i r e a s u r p l u s o f a s s i m i l a t e s , i . e . W( a -1) i n order to produce f l o w e r s , f r u i t s , e t c . from an a s s i m i l a t o r y apparatus of f i n i t e s i z e . Growth a n a l y s i s has c l a s s i c a l l y been done by computation from averages of d i s c r e t e h a r v e s t s taken a t s e v e r a l i n t e r v a l s of time d u r i n g the growth d u r a t i o n o f the p l a n t . As a consequence, r e s u l t i n g c a l c u l a t i o n s have been i n a c c u r a t e and i m p r e c i s e . A c u r v e - f i t t i n g approach u s i n g stepwise m u l t i p l e r e g r e s s i o n s (Radford, 1967) has been shown to be more a c c u r a t e . But t h i s was very l a b o r i o u s and time-consuming p r i o r to the a v a i l a b i l i t y of computing f a c i l i t i e s and computer programs such as those o f Hunt and Parsons (1974; 1977). 18 The technique o f growth a n a l y s i s , s i n c e i t s i n c e p t i o n more than 50 years ago, has been used to study growth c h a r a c t e r i s t i c s and to q u a n t i f y the accumulation of dry matter of f i e l d crops*grown under v a r i o u s c o n d i t i o n s . R o l l e r , N y q u i s t and Chorush (1970) , i n s t u d y i n g components of dry matter accumulation i n f i e l d soybeans, found t h a t RGR of i n d i v i d u a l p l a n t f r a c t i o n s ( l e a f , stem, etc.) s t e a d i l y decreased as the p l a n t matured. At any given time the most r e c e n t l y i n i t i a t e d p l a n t f r a c t i o n had the g r e a t e s t RGR. T o t a l above-ground RGR d e c l i n e d u n t i l e a r l y pod formation when i t peaked c o n c u r r e n t l y w i t h an i n c r e a s e i n NAR. They i n t e r p r e t e d the i n c r e a s e i n NAR as a response of the p h o t o s y n t h e t i c apparatus to an i n c r e a s e d demand f o r a s s i m i l a t e s due t o the r a p i d growth of the seed f r a c t i o n . B u t t e r y (1969) s t u d i e d the e f f e c t s of p o p u l a t i o n d e n s i t y and f e r t i l i z e r a p p l i c a t i o n on f i e l d - g r o w n soybeans, and found t h a t , r e g a r d l e s s of treatment, there was a d e c l i n e i n NAR and RGR towards m a t u r i t y . The d e c l i n e was a t t r i b u t e d p r i m a r i l y to i n c r e a s i n g l e a f i n e s s . Thome (19 60) r e p o r t e d t h a t , under c o n t r o l l e d e n v i -ronment, NAR o f sugar-beet, p o t a t o , and b a r l e y f e l l approximately l i n e a r l y w i t h time. During 5 weeks, NAR of sugar-beet and potato decreased by 20 and 50 p e r c e n t r e s p e c t i v e l y . NAR of b a r l e y remained approximately constant f o r 4 weeks but was h a l v e d d u r i n g the subsequent weeks. RGR, R L and LAR f e l l w i t h time a t s i m i l a r r a t e s f o r a l l three crop s p e c i e s . 19 Some s t r e s s f a c t o r s have been shown t o have c o n t r a s t i n g i n f l u e n c e s on growth parameters. For example, L a s t (1962) rep o r t e d t h a t the changes i n r o o t development and l e a f area caused by mildew(Erysiphe graminis D.C.) on b a r l e y were a s s o c i a t e d w i t h s i m i l a r decreases i n NAR. From 12 to 68 2 days a f t e r i n o c u l a t i o n the mean NAR was 226.6 mg/dm /w 2 i n mildew-free c o n t r o l s and 166.0 mg/dm /w i n the i n o c u l a t e d s e r i e s . In young tomato p l a n t s s u b j e c t e d to w i l t i n g treatments o f s h o r t d u r a t i o n , Gates (1955) r e p o r t e d t h a t NAR and RGR was reduced d u r i n g the p e r i o d o f w i l t i n g but the growth parameters were g r e a t e r than f o r c o n t r o l p l a n t s upon r e w a t e r i n g . During w i l t i n g , h i g h e r stem weight r a t i o s and lower l e a f weight r a t i o s developed than i n the c o n t r o l , whereas a f t e r w i l t i n g , l e a f weight r a t i o s were hi g h e r than stem weight r a t i o s . However, there was no i n d i c a t i o n t h a t the recovery e f f e c t was complete at the f i n a l h a r v e s t . Gates (1955) i n t e r p r e t e d these treatment e f f e c t s as a tendency towards senecence d u r i n g w i l t i n g and a r e t u r n to a more j u v e n i l e c o n d i t i o n upon rewatering. He concluded t h a t the changes i n weight r a t i o s were due to m o d i f i c a t i o n s o f the normal p a t t e r n o f t r a n s l o c a t i o n between p l a n t p a r t s . Tsiung (1978) , i n a study on the response o f mung-bean to sowing dates i n the Malaysian Borneo s t a t e of Sarawak (4°07' N; 113°57*E) , a p p l i e d the technique o f growth a n a l y s i s t o c h a r a c t e r i z e the marked growth d i f f e r e n c e s among the sowing dates. A P h i l i p p i n e v a r i e t y , CES-55 used 20 i n t h i s study, was sown at the b e g i n n i n g of March, May, J u l y , August and September, 1976. Although there were marked d i f f e r e n c e s i n p l a n t growth, the phenology was s i m i l a r f o r a l l p l a n t i n g d a t e s : f l o w e r i n g a t 33 days a f t e r sowing, r i p e n i n g of bean pods a t 51 days and m a t u r i t y by day 70. Dry matter accumulation was a l s o s i m i l a r slow d u r i n g the f i r s t 20 days, f o l l o w e d by a r a p i d i n c r e a s e once f l o w e r i n g and p o d - s e t t i n g commenced, a t t a i n i n g a maximum at day 60. Tsiung (1978) found out t h a t the p a t t e r n o f changes i n RGR i n a l l sowing dates was c h a r a c t e r i z e d by an i n c r e a s e from day 15 to a peak at day 25 f o l l o w e d by a r a p i d , smooth decrease t h e r e a f t e r , a t t a i n i n g a n e g a t i v e value a t day 65. NAR behave s i m i l a r l y except t h a t from day 25 to 55 the decrease was at a slower r a t e but dropped to a n e g a t i v e v a l u e a l s o a t day 65. The decrease i n RGR was a t t r i b u t e d to a f a s t e r r a t e o f d e c l i n e i n LAR (56%) than i n NAR (36%). As f a r as I can determine, there i s no r e p o r t on the a p p l i c a t i o n of growth a n a l y s i s on p l a n t s grown under s t r e s s of a l l e l o p a t h y . 21 MATERIALS AND METHODS The experiments were conducted i n the greenhouse of the Department of P l a n t S c i e n c e , U n i v e r s i t y of B r i t i s h Columbia, at a l a t i t u d e o f 49°16'N. The temperature regime was e s t a b l i s h e d such t h a t minima ranged between 20° and 22°C and maxima between 28° and 30°C. During w i n t e r months supplementary l i g h t i n g was p r o v i d e d f o r 12 hours per day by means of f i v e 400-watt high p r e s s u r e Sodium-vapor (Lucalox) lamps arranged i n a row 1.6 m above the c e n t e r o f the four-row pot arrangement. The rows of the pots were spaced such t h a t a l l the pots were l o c a t e d w i t h i n the 1.2m-wide area where the l i g h t i n t e n s i t y was uniform. The i l l u m i n a t i o n measured 140 cm below the l i g h t s at 16:00 on a o v e r c a s t day was 518 - 13 and 5 34 - 17 f o o t - c a n d l e s on the n o r t h and south s i d e rows r e s p e c t i v e l y . The i n t e n s i t i e s over the two middle rows were 620 - 15 and 610 - 15 f o o t - c a n d l e s . Outside the greenhouse at the time o f the above measurements the i n t e n -s i t y was 156 f o o t - c a n d l e s . The r e l a t i v e humidity f l u c t u a t e d between an average maximum o f 80% and an average minimum o f 55%. General Management 3 A l l the p l a n t s were grown i n 4 800 cm growing medium. The r i v e r sand used i n Expts. 1 and 2 was washed thoroughly w i t h water u n t i l the washings were c l e a r . The 22 s o i l used i n a l l experiments was taken from the steam-s t e r i l i z e d p o t t i n g s o i l supply i n the greenhouse. Gravels and other matters l a r g e r than 1 cm diameter were screened out. Added amounts of f e r t i l i z e r s were shovel-mixed i n measured batches corresponding t o one r e p l i c a t i o n . In Experiments 1 and 2, the mungbean c u l t i v a r used was MG50-10a (green-seeded). S i n c e an e a r l i e r p l a n t i n g o f t h i s c u l t i v a r had shown wide v a r i a b i l i t y (some were purple-based and tend t o be v i n y ) , heavy seeding r a t e s were used and o f f - t y p e s were rogued out 10 days a f t e r emergence. Yellow-seeded MG50-10a was used i n Experiments 3a and 3b. Again, two types were observed: d u l l y e l l o w and g l o s s y y e l l o w . The l a t t e r were s e l e c t e d and were observed to be uniform. Maintenance of a l l experiments c o n s i s t e d o f d a i l y i n s p e c t i o n f o r moisture s t a t u s o f the p o t s , temperature and humidity extremes, and occurence o f p e s t s . The s o i l was not allowed to dry up on the s u r f a c e nor was i t allowed t o become water-logged. A hygrograph and thermograph p l a c e d near the center of the greenhouse monitored the d a i l y humidity and temperature ranges. The d e s i r e d temperature range was maintained by means of the auto-matic temperature c o n t r o l s o f the greenhouse. Humidity was kept above 50% by p e r i o d i c s p r i n k l i n g of water around the experimental area. The p l a n t s were sprayed with p r o p a r g i t e 30WP at 1.2 5 g / l i t e r , a m i t i c i d e , as mite i n f e s t a t i o n s were observed a f t e r pod s e t . Regular fumigations of the greenhouse complex were s u f f i c i e n t to c o n t r o l o t h e r 23 pests, p a r t i c u l a r l y the greenhouse whitefly. Experiment 1 The objective of this experiment was to determine whether materials leached from the roots of mungbean plants growing i n sand culture during t h e i r growing period and transferred continuously to pots of steam-sterilized s o i l would accumulate and influence the growth of mung-bean plants sown subsequently i n the re c i p i e n t s o i l . The experimental set-up was composed of: a) a nutrient solution container with d i s t r i b u t i o n tubing; b) donor pots with sand as the rooting medium; c) recipient pots with f e r t i l i z e d , steam-sterilized potting s o i l as the rooting medium. The pots and nutrient supply were arranged as a s t a i r s t e p system i n which each donor pot leached into a r e c i p i e n t pot by means of tubing. The general arrangement i s shown in F i g . l a and in close-up i n F i g . 1 b. The d i s t r i b u t i o n and regulation of the nutrient flow to the i n d i v i d u a l pots were accomplished in the following manner: PVC black tubing (1.27 cm ID) of appropriate length was connected at one end v i a a check valve to the nutrient solution container; the other end was connected v i a a p l a s t i c pipe reducer to a f l e x i b l e tygon tube (0.64 cm OD) open at the other end. This tygon tube was long enough to serve as a "standpipe", 24 25 to balance the pressure head of the n u t r i e n t s o l u t i o n i n the container and to avoid the problem of a i r trapped w i t h i n the PVC tubing. Along the length of the PVC tube, f i n e PVC leader tubes (1.2mm ID) were connected, v i a brass i n s e r t c o u p l e r s , t o d e l i v e r the n u t r i e n t s o l u t i o n drop by drop to the donor pots. The o u t l e t ends of the leader tubes were f i x e d at a height to d e l i v e r 6 drops per minute to each donor pot. Two leader tubes were provided per pot to d i s t r i b u t e the moisture more evenly on the surface of the r o o t i n g medium. Glazed ceramic pots (17 cm dia) w i t h a d r a i n hole (2.54 cm dia) on the s i d e near the bottom were used f o r both donor and r e c i p i e n t pots . A s e m i - f l e x i b l e poly-propylene tube (0.64 cm OD) of appropriate length was f i t t e d t i g h t l y at one end i n t o a hole d r i l l e d through a rubber stopper i n s e r t e d i n t o the hole i n the donor pot. The other end of t h i s tube was plugged. Two leader tubes were connected v i a brass i n s e r t couplers near the plugged end, t o d e l i v e r leachate to each r e c i p i e n t pot, as shown i n F i g . 1 b. N u t r i e n t s o l u t i o n . N u t r i e n t s o l u t i o n No. 1 as described i n C a l i f o r n i a A g r i . Expt. Sta. C i r . 347 (Hoagland and Arnon, 1950) was allowed to flow through each experimental u n i t . The flow from the n u t r i e n t supply was regulated such that the leachate s o l u t i o n s were d e l i v e r e d from the donor to the r e c i p i e n t pots at a r a t e which avoided excessive flow through the r e c i p i e n t pots to waste. The amount of s o l u t i o n that drained from the 26 r e c i p i e n t pot was minimal and was not r e c i r c u l a t e d back to the system. Donor p o t s . I t was expected t h a t the low absor-bin g c a p a c i t y of sand would allow the maximum t r a n s f e r to the r e c i p i e n t pots of whatever exudate was r e l e a s e d from the p l a n t s i n the donor p o t s . However, at the c o n c l u s i o n o f each p e r i o d of l e a c h a t e t r a n s f e r (see s e c t i o n on R e c i p i e n t p o t s , below) the donor pots were di s c o n n e c t e d and f l u s h e d with two volumes o f water to wash out remaining exudates. The washings were saved and used to water the r e c i p i e n t pots u n t i l consumed. The donor pots were seeded to a f i n a l stand of 4 p l a n t s / p o t except f o r the c o n t r o l s . R e c i p i e n t p o t s . The use o f s t e a m - s t e r i l i z e d s o i l minimized the i n f l u e n c e o f microorganisms and excluded contamination by weed seeds which might have germinated and i n f l u e n c e the r e s u l t . Seeding t o a f i n a l stand of 4 p l a n t s per pot was done 7 days a f t e r the end of the l e a c h i n g p e r i o d , which approximates the time i n t e r v a l between s u c c e s s i v e p l a n t i n g s i n the f i e l d . Slow-release f e r t i l i z e r (Osmocote, 14-14-14 NPK) was added at the r a t e 3 of 74g/dm to i n s u r e an adequate supply o f n u t r i e n t s , s i n c e the r e c i p i e n t p l a n t s were watered from the tap a f t e r the s t o r e d l e a c h a t e s had been consumed. Two p e r i o d s o f l e a c h a t e accumulation were i n v e s t i g a t e d . In one, l e a c h a t e s were t r a n s f e r r e d up to the end o f the v e g e t a t i v e stage o f growth of the donor p l a n t s (about 28 days). In the o t h e r , l e a c h a t e t r a n s f e r continued to m a t u r i t y (about 70 days). A separate s e t w i t h no p l a n t s i n the donor pots served as the c o n t r o l , f o r each l e a c h i n g p e r i o d . 27 Experimental d e s i g n . Four treatments c o n s i s t i n g of two p e r i o d s of l e a c h a t e accumulation and one u n t r e a t e d c o n t r o l f o r each p e r i o d comprised a r e p l i c a t e . A treatment c o n s i s t e d o f two p o t s : donor and r e c i p i e n t . There were f i v e r e p l i c a t i o n s f o r a t o t a l of 40 pots i n a randomized complete b l o c k d e s i g n . Experiment 2. Th i s experiment i n v o l v e d the l e a c h i n g o f decom-p o s i t i o n products of r o o t s and l e a v e s . The same experimental system was used as i n the f i r s t experiment, except t h a t the donor pots contained decomposing r e s i d u e s which leached i n t o r e c i p i e n t p o t s . The p l a n t s of the donor pots i n Experiment 1 were grown to m a t u r i t y a f t e r which the tops were c l i p p e d o f f . These served as the r o o t r e s i d u e donor p o t s . The leaves from two o f the pots i n a r e p l i c a t e were pooled and i n c o r p o r a t e d i n t o one of the blank donor pots of the same r e p l i c a t e . These pots served as the l e a f r e s i d u e donor p o t s . The donor pots thus c o n s i s t e d o f two pots with r o o t r e s i d u e s , one pot w i t h l e a f r e s i d u e s and one c o n t i n u i n g c o n t r o l pot, per b l o c k . A l l the p l a n t r e s i d u e s i n these pots were allowed t o decompose f o r one week b e f o r e l e a c h i n g . Leaching was done f o r t h i r t y days by w a t e r i n g the donor pots from the tap j u s t enough to soak the r e c i p i e n t pots every o t h e r day. The c o n t r o l 28 donor pot was t r e a t e d i n the same way as the pots w i t h r e s i d u e s . The r e c i p i e n t pots i n t h i s experiment had the same s o i l source as i n Experiment 1. The same r a t e o f f e r t i l i z e r was a l s o added and seeding t o a f i n a l stand o f 4 p l a n t s / p o t was done immediately a f t e r the 30-day l e a c h i n g p e r i o d . Again, a randomized complete b l o c k design was used. Experiment 3a. The o b j e c t i v e o f t h i s experiment was to determine whether d i r e c t c o n t a c t between the r o o t s of a subsequent mungbean crop and the r o o t and l e a f r e s i d u e s from a p r e v i o u s mungbean crop grown i n s o i l was a requirement i n o r d e r f o r the second crop to be a f f e c t e d . I t was conducted i n two stages. T h i s approach s i m u l a t e d the s e q u e n t i a l cropping as p r a c t i c e d i n the f i e l d . E s t a b l i s h m e n t of f i r s t crop. Pots o f s o i l with f e r t i l i z e r added a t the same rate>as i n Experiment 1 were seeded t o a f i n a l stand o f 4 p l a n t s / p o t . (Pots were thinned 5 days a f t e r sowing). The same number of pots o f s o i l were a l s o prepared along w i t h the cropped s o i l and l a i d f a l l o w f o r the d u r a t i o n of the f i r s t crop. The p l a n t s were grown to m a t u r i t y , i . e . , when most o f the pods had turned b l a c k , a f t e r which the leaves were c o l l e c t e d , 29 d r i e d , ground, and i n c o r p o r a t e d back i n t o s o i l o f the corresponding p o t s , which c o n t a i n e d the r o o t r e s i d u e s in s i t u . Second crop. The pots o f cropped s o i l were d i v i d e d t o p r o v i d e two " i n c u b a t i o n " p e r i o d s . Seeding to a f i n a l stand of 4 p l a n t s / p o t was done a f t e r one-week and a f t e r three-week i n c u b a t i o n p e r i o d s . A corresponding number of f a l l o w pots were a l s o seeded f o r each i n c u b a t i o n p e r i o d to serve as c o n t r o l s . The growth o f the p l a n t s was monitored by sampling at f o u r s t a g e s : 1) 14 days a f t e r emergence (DAE), when the f i r s t t r i f o l i a t e leaves had expanded; 2) 28 DAE, f l o w e r i n g stage; 3) 42 DAE, p o d - f i l l i n g stage; 4) 73 DAE, m a t u r i t y — when 70% of the pods had turned b l a c k . Experimental d e s i g n . The experiment c o n s i s t e d of 4 treatments, 4 sampling d a t e s , and 5 r e p l i c a t i o n s , a t o t a l o f 80 p o t s . Since the treatments were composed o f two groups of p l a n t s w i t h a two-week age d i f f e r e n c e , i t was thought t h a t a random arrangement o f a l l the pots would have been disadvantageous to the younger p l a n t s because of p o s s i b l e shading by the o l d e r p l a n t s . A s p l i t -p l o t d e sign w i t h treatments as the main p l o t i n s t r i p s and sampling dates as s u b p l o t s was t h e r e f o r e used. The vacant spaces c r e a t e d as sampling was done were f i l l e d by " f i l l e r p o ts" of p l a n t s seeded at the same time as the experimental p l a n t s . T h i s was done i n order 30 to minimize the edge-effect of the vacant spaces created by harvesting. Border pots of plants were also placed around the whole experimental area. Experiment 3b. In th i s experiment I studied the growth of a second mungbean crop, comparing s o i l which contained both root and leaf residues against s o i l which contained either root residues alone or leaf residues alone. In th i s way the leaf residue e f f e c t was separated from the e f f e c t of root residue. The same general procedure was followed as i n Experiment 3a, i . e . , the establishment of the f i r s t crop followed by the second crop. The treatments were established by dividing the number of pots of cropped s o i l and fallow s o i l into halves. The le a f residues from one half of the cropped pots were incorporated into the corresponding number of pots of the fallow (control) s o i l . This made a t o t a l of 20 pots with roots plus leaves, 20 pots with roots alone, 20 pots with leaves alone, and 20 pots without residues. The second crop was seeded after 12 days' incubation of the plant residues. The pots were arranged i n a randomized complete block design consisting of 4 treatments, 4 sampling dates and 5 r e p l i c a t e s , a t o t a l of 80 pots. 31 C o l l e c t i o n o f data For a l l h a r v e s t s , the p l a n t s were cut o f f at the crown o f the r o o t s . Leaves ( i n c l u d i n g p e t i o l e s ) were separated from the stems, and l e a f area measurements were made immediately w i t h a Hayashi Denko AAM-5 automatic p h o t o e l e c t r i c i n t e g r a t i n g area meter. Where r o o t weight was recorded, r o o t s were se p a r a t e d from s o i l by means o f 5-mm screenwire mesh and subsequent washing i n water w i t h the a i d o f a f i n e k i t c h e n s i e v e . Washed r o o t s were b l o t t e d i n paper towels b e f o r e bagging. Leaves, r o o t s , stems, and pods from each pot were bagged and l a b e l l e d s e p a r a t e l y b e f o r e oven d r y i n g at 70°C f o r a t l e a s t 48 hours. The oven-dried samples were co o l e d to room temperature s e a l e d i n p l a s t i c bags p r i o r t o weighing. Each p l a n t p a r t (stem, l e a v e s , etc.) was weighed s e p a r a t e l y and weights were expressed i n g/pot of 4 p l a n t s . The l e a f a r e a rea d i n g i n 2 2 2 cm was converted i n t o dm and was expressed as dm /pot of 4 p l a n t s . For Experiments 3a and 3b, SLA, LWR, LAR values were c a l c u l a t e d by means of a desk c a l c u l a t o r and were tab-u l a t e d together w i t h l e a f area (L) , l e a f weight(W^) , stem weight (W g), pod weight (W p), r o o t weight (WR) and t o t a l p l a n t dry weight (W), which i s the sum of a l l component weights. These values were s u b j e c t e d t o a n a l y s i s o f v a r i a n c e through the f a c i l i t i e s o f the UBC Computing Centre. No s t a t i s t i c a l a n a l y s i s were done on the d a t a gathered from Experiments 1 and 2 s i n c e the f a m i l i a r symptoms of the e f f e c t o f 32 a previous crop were not observed and the p l a n t s i n the r e c e p t o r pots were uniform i n s i z e and appearance. C a l c u l a t i o n s of the growth parameters R, R L, E and a f o r Experiments 3a and 3b were f i r s t done w i t h the t r a d i t i o n a l method d e s c r i b e d by Evans (1972) and Sestak e t a l . (1971). But s i n c e i t was d i f f i c u l t to a s c e r t a i n from the c a l c u l a t e d values the a c t u a l p a t t e r n of growth because of l a c k of sampling p o i n t s along the growth curve, the c u r v e - f i t t i n g approach (mentioned on page 17) was r e s o r t e d t o . Cubic polynomial equations (see Appendix 1) were f i t t e d t o the changes i n mean t o t a l dry weight (W) and l e a f area (L) w i t h sampling time ( t ) . These can be r e p r e s e n t e d (Sestak e t a l . , 1971) as the f o l l o w i n g : W = f x ( t ) = a + b t + c t 2 + d t 3 L = f 2 ( t ) = a' + b ' t + c ' t 2 + d ' t 3 from which R, R^, E and a can be d e r i v e d thus: R = d f l ( t ) dt f x ( t ) d f 2 ( t ) d t f 2 ( t ) E = d f l ( t ) dt f 2 ( t ) a = R/R. 33 In g e n e r a t i n g the polynomial equations, a sm a l l dummy number was used at t=0 i n both Experiments 3a and 3b. F i g u r e s 13 and 14 show the f i t t e d curves f o r W and L. The a c t u a l and f i t t e d v alues f o r Experiment 3a are pres e n t e d i n Appendices 2 and 3. In Experiment 3b the f i t t e d v a lues were e x a c t l y the same as the a c t u a l values s i n c e there were onl y three sampling p o i n t s . The means o f the a c t u a l values f o r W, WL, WR, Wg, Wp, and L f o r Experiment 3a and 3b are pres e n t e d i n Appendices 4 and 5. For convenience i n p r e s e n t a t i o n , the u n i t s of the f o l l o w i n g parameters were changed: R g.g ^".day ^ t o mg.g ^".day ^ -2 -1 -2 -1 E g.dm .day to mg.dm .day dm2.dm 2.day ^ t o cm2.dm 2.day * C a l c u l a t i o n o f o was based on the o r i g i n a l u n i t s o f R and P^. 34 RESULTS The c u r r e n t use o f the term a l l e l o p a t h y r e f e r s to the harmful e f f e c t s o f h i g h e r p l a n t s o f one s p e c i e s (the donor) on the germination, growth, o r development of p l a n t s of another (receptor) s p e c i e s (Putnam and Duke, 1978). In the f o l l o w i n g d i s c u s s i o n s the use of the term i s extended to apply to the d e t r i m e n t a l e f f e c t s of a p r e v i o u s crop (the donor) on the growth and develop-ment o f the succeeding crop (receptor) o f the same s p e c i e s . Leachate t r a n s f e r . Experiment 1 was undertaken i n an attempt to demonstrate whether w a t e r - s o l u b l e t o x i n s are exuded from h e a l t h y i n t a c t r o o t s of mungbean, accumulate i n r h i z o s p h e r e , and may be t r a n s f e r r e d and made to accumulate i n a s o i l medium without l o s s of t o x i c i t y . The r e c e p t o r p l a n t s d i d not show any obvious, v i s i b l e d i f f e r e n c e s from the c o n t r o l s from the time o f germination to m a t u r i t y . The weight o f tops and l e a f areas o f the r e c e p t o r p l a n t s are presented i n Tables 1 and 2 r e s p e c t i v e l y . I n s p e c t i o n of these data r e v e a l s no s i g n i f i c a n t e f f e c t s of treatment per se, and no d i f f e r e n c e s between the 28-day and 70-day l e a c h i n g p e r i o d s . 35 Table 1.. Top weight (g/pot of 4 plants) of mungbeans grown i n s o i l that had received leachates from mungbeans growing on sand culture. Harvested at maturity. (70 DAE) Treatment Replication Mean 1. 28-day leaching 22.0 30.4 23.2 33.6 31.6 28.2 2. Control for 33.6 31.6 22.0 30.4 21.2 27.8 Treat. No. 1 3. 70-day leaching 33.2 28.0 24.0 32.0 24.8 28.4 4. Control for 31.6 23.4 22.1 30.3 33.4 28.2 Treat. No.3 2 Table 2. Leaf area (dm /pot of 4 plants) of mungbeans grown in s o i l that had received leachates from mungbeans growing on sand culture. Harvested at maturity. (70 DAE). Treatment R e p l i c a t i o n Mean 1. 28-day leaching 20.9 27.9 21.9 30.4 27.1 25.6 2. Control for 29.4 27.8 19.8 26.4 18.8 24.4 Treat. No. 1 3. 70-day leaching 28.9 24.9 18.7 28.2 20.2 24.2 4. Control for 28.3 20.9 18.9 27.6 30.2 25.2 Treat. No. 3 36 Experiment 2 attempted t o show i f there are wa t e r - s o l u b l e p h y t o t o x i n s t h a t would l e a c h out of decom-posing r e s i d u e s a f t e r death. The donor p l a n t s which were grown to m a t u r i t y i n Experiment 1 were used i n t h i s experiment. T h e i r r o o t or l e a f r e s i d u e s were allowed to decompose i n the o r i g i n a l sand medium and le a c h a t e s were t r a n s f e r r e d i n t o s o i l i n which the r e c e p t o r p l a n t s were grown. Tables 3, 4 and 5 show top weights, l e a f areas and h e i g h t s r e s p e c t i v e l y o f the r e c e p t o r p l a n t s h a r v e s t e d a t the f l o w e r i n g stage, as a f f e c t e d by l e a c h a t e accumulation from decomposing leaves and r o o t s . As i n Experiment 1, there were no v i s i b l e d i f f e r e n c e s between the t r e a t e d p l a n t s and the c o n t r o l s , from the time o f emergence to h a r v e s t date. S i m i l a r l y , the data r e v e a l no s i g n i f i c a n t e f f e c t s o f treatment. However, i t i s of i n t e r e s t to note that the data suggest a g r e a t e r e f f e c t of l e a f r a t h e r than r o o t r e s i d u e s . Table 3. Top weight (g/pot o f 4 p l a n t s ) o f mungbeans grown i n s o i l t h a t had r e c e i v e d l e a c h a t e s of decomposing roo t s and l e a v e s . Harvested a t f l o w e r i n g stage. (35 DAE) Treatment ; R e p l i c a t i o n Mean 1 2 3 4 5 1. Decomposing r o o t 11. 0 15.2 11.6 16. 8 15. 8 14. 1 2. " leaves 16. 8 15. 8 11.0 15. 2 10. 6 13. 9 3. C o n t r o l 16. 6 14.0 12.0 16. 0 12. 4 14. 2 37 Table 4. Leaf area (dm /pot o f 4 p l a n t s ) o f mungbeans grown i n s o i l t h a t had r e c e i v e d l e a c h a t e s o f decomposing ro o t s and l e a v e s . Harvested a t f l o w e r i n g stage. (35 D A E ) . Treatment R e p l i c a t i o n Mean 1 2 3 4 5 1. Decomposing roo t 18. 6 16.4 17.1 18. 2 18. 8 17. 8 2. " leaves 21. 4 19.6 14.2 18. 2 14. 2 17. 5 3. C o n t r o l 19. 6 16.2 13.9 19. 8 20. 4 18. 0 Table 5 . Average p l a n t h e i g h t (cm) of mungbeans grown i n s o i l t h a t had r e c e i v e d l e a c h a t e s o f decomposing roo t s and l e a v e s . Harvested a t f l o w e r i n g stage. (35 D A E ) . Treatment R e p l i c a t i o n Mean 1. Decomposing r o o t 38. 4 38. 1 38. 7 36. 9 37. 8 38. 0 2. " leaves 41. 0 41. 3 34. 3 37. 8 32. 4 37. 4 3. C o n t r o l 42. 0 37. 8 36. 3 40. 6 37. 5 38. 8 38 S e q u e n t i a l cropping. S i n c e none of the l e a c h a t e - t r a n s f e r experiments showed the t y p i c a l symptoms of the e f f e c t of the donor on the r e c e p t o r p l a n t s , i t was deduced t h a t the problem does not i n v o l v e a mere s t r a i g h t f o r w a r d r e l e a s e of phytotoxins which can be e a s i l y leached from the growing medium. With t h i s i n view, I attempted to simulate as c l o s e l y as p o s s i b l e the c o n d i t i o n s as p r a c t i c e d i n f i e l d c r opping. Where the problem i s observed, the second crop i s e s t a b l i s h e d i n quick s u c c e s s i o n to the f i r s t , w i t h the leaves and r o o t r e s i d u e s of the f i r s t crop worked i n t o the seedbed. Experiment 3a was p r i m a r i l y designed to compare the growth of mungbeans i n f a l l o w s o i l and cropped s o i l into which were i n c o r p o r a t e d r o o t and l e a f r e s i d u e s of a previous mungbean crop. A second o b j e c t i v e was to f i n d out i f the l e n g t h of i n c u b a t i o n o f the r e s i d u e had an i n f l u e n c e on the problem. Germination i n both cropped and f a l l o w s o i l was uniform and occured e s s e n t i a l l y a t the same time. But the treatments showed observable d i f f e r e n c e s immediately a f t e r emergence. Those p l a n t s grown i n cropped s o i l showed the t y p i c a l symptoms observed i n the f i e l d . Thus, expansion of the c o t y l e d o n a r y leaves was i n h i b i t e d ( F i g s . 2 and 3), growth was stunted ( F i g s . 4 and 5) and r o o t development was s e v e r e l y reduced ( F i g s . 6 and 7) i n a l l the p l a n t s 39 F i g u r e 2. Reduced s i z e of mungbean s e e d l i n g s a t 7 DAE grown i n cropped s o i l w ith r o o t and l e a f r e s i d u e i n c o r p o r a t e d and incubated f o r one week. Note the e a r l y development of the f i r s t t r i f o l i a t e l e a v e s i n the no-residue s o i l . F i g u r e 3. Reduced cotyledonary l e a f expansion o f mungbean grown i n the r e s i d u e t r e a t e d s o i l . 14 DAE. 40 Fi g u r e 4. Stunted growth of mungbean grown i n cropped s o i l w i t h r o o t and l e a f r e s i d u e i n c o r p o r a t e d and incubated f o r one week. 14 DAE. F i g u r e 5. The same treatment as i n F i g u r e 4 a t 28 DAE. G r i d l i n e s i n the above photographs are 15.24 cm a p a r t . 41 F i g u r e 6 . Root development of mungbean shown i n F i g u r e 4. F i g u r e 7. Root development o f mungbean a t 28 DAE o f the same treatment as i n F i g u r e 4. 42 of the cropped s o i l . Examination of the s e e d l i n g s from cropped s o i l about 3 days a f t e r emergence showed that the base of the stem was t h i c k e n e d and c u r l e d and the tap r o o t d i d not develop ( F i g s . 8 and 9 ) as compared t o normal s e e d l i n g s of the same age ( F i g . 1 0 ) . T o t a l dry weights, component weights ( l e a v e s , stems, r o o t s , and pods), l e a f areas, LAR's, LWR's and SLR's were s u b j e c t e d to a n a l y s i s o f v a r i a n c e . Comparisons and c o n t r a s t s of treatment e f f e c t s were done i n the f o l l o w i n g manner: a) one- vs 3-week i n c u b a t i o n X l = 1 / 2 ( T 1 + T 2) vs x 2 = 1 / 2 ( T 3 + T 4 ) ; b) w i t h vs without r e s i d u e x 3 = 1 / 2 ( T x + T 3) vs x 4 = 1 / 2 ( T 2 + T 4 ) ; where T^ = Treatment with 1-week i n c u b a t i o n o f r e s i d u e s ; T 2 = C o n t r o l f o r T^-T^ = Treatment wi t h 3-week i n c u b a t i o n of r e s i d u e s ; T 4 = C o n t r o l f o r T 3 . Table 6 summarizes the analyses o f v a r i a n c e done on the component and t o t a l dry weights, and c o n t r a s t s the e f f e c t s of the treatments (with vs without r e s i d u e s ) and the e f f e c t s of the treatments (one- vs three-week i n c u b a t i o n p e r i o d ) . O b v i o u s l y , the r o o t and l e a f r e s i d u e s s e v e r e l y reduce the component weights and hence the t o t a l dry weights of the r e c e p t o r p l a n t s . The e x t e n t of the r e d u c t i o n s caused by the i n c o r p o r a t i o n o f the r e s i d u e s i s presented 4 3 FALLOU) SOIL 6 P4HS AFTIR SefPW6 O8-/5 - 7& CKorpeo sen. IV/T-H 3 UlFFftf IUOH RAJ ION OF PLANT Resieue & DAHS AFTl*. IttDfAi* 08- /S -78 Figure 8 . The e f f e c t of root and le a f residues, incorporated and incubated for three weeks into the s o i l , on the development of mungbean seedlings. 3 DAE (6 days afte r seeding). Note the s l i g h t l y thickened and curled basal portion of the hypocotyl i n contrast to the untreated s o i l (fallow). . IVIT-H 3 I'trm mo J RATIO A OF PLANT ft 7 DAY* AFTf* Figure 9 . The same treatment as i n Figure 8 at 7 DAE. Note the abnormal development of the primary root. Above magnification i s twice that of F i g . 8 . 44 Figure 10. Normal seedling development of mungbean at 7 DAE. Magnification i s 1:4. 45 i n Figure 11. The l i n e a r regression l i n e s are highly s i g n i f i c a n t l y d i f f e r e n t (Table 6 ) . The primary data are presented i n Appendix 4. Table 6. Summary of contrasts between effects of the treatments: with vs without residue, and the effects of the treatments: one- vs three-week incubation period. Variables with vs without residue one- vs three-week incubation incubation X residue in t e r a c t i o n Total dry weight ** ** ns leaf area ** ** ns leaf weight ** ** ns stem weight ** ns * root weight ** ** ns pod weight ** * ns LAR * ns ns LWR * ns ns SLA ns ns ns ** S t a t i s t i c a l l y * S t a t i s t i c a l l y s i g n i f i c a n t s i g n i f i c a n t at 1% l e v e l , at 5% l e v e l . ns Non-significant Incubation of the residues for up to three weeks appears to enhance i t s deleterious e f f e c t s on the Figure 1 1 . Growth of mungbean i n cropped s o i l with leaf and root residues of the previous crop incorporated ( • ), and i n s o i l that was l a i d fallow, without residues ( o ). Total dry weights (W), l e a f (WL), stem (Wg), root (WR), and pod (Wp) weights are expressed i n g/pot of 4 plants. Leaf 2 area (L) i s expressed i n dm /pot of 4 plants. Each point represents the mean across incubation periods. For each parameter, the slopes of the l i n e a r regressi l i n e s are highly s i g n i f i c a n t l y d i f f e r e n t . 47 DAYS A F T E R E M E R G E N C € 48 accumulation o f dry matter i n a l l p a r t s of the p l a n t and on l e a f area (Table 7 ). The o n l y e x c e p t i o n i s stem weight (Table 6). T h i s l a c k of e f f e c t o f i n c u b a t i o n p e r i o d on stem weight probably accounts f o r the s o l e s i g n i f i c a n t i n t e r a c t i o n between i n c u b a t i o n p e r i o d and r e s i d u e (Table 6 ) . Table 7. Comparison o f means across a l l h a r v e s t s f o r 1-week and 3-week i n c u b a t i o n p e r i o d s w i t h l e a f and r o o t r e s i d u e s . Incubation p e r i o d one week three weeks V a r i a b l e s Treated C o n t r o l Treated C o n t r o l (g/pot of 4 p l a n t s ) T o t a l dry weight 9.20 20.80 5.03 18.76 l e a f weight 3.54 7.53 1.66 6.69 stem weight 2.17 5.08 1.07 5.35 r o o t weight 0.93 2.12 0.46 1. 89 pod weight 2.57 6.08 1.84 4. 84 2 (dm /pot of 4 p l a n t s ) l e a f area 10.51 20.80 5.08 20.45 Since the a n a l y s i s o f v a r i a n c e (Table 6 ) r e v e a l e d no major i n t e r a c t i o n s between the two treatment s e t s , the mean values of the d e r i v e d growth parameters f o r the one- and three-week i n c u b a t i o n s were i n i t i a l l y compared 49 t o t h e means o f t h e c o r r e s p o n d i n g c o n t r o l s ( F i g . 1 2 ) . The e f f e c t s o f r e s i d u e s a r e c l e a r l y shown, w i t h t h e m a j o r d i f f e r e n c e s o c c u r i n g d u r i n g t h e e a r l y s t a g e s o f g r o w t h . The e x c e p t i o n i s t h e e f f e c t on r e l a t i v e l e a f a r e a g r o w t h r a t e , i n w h i c h t h e r e i s a c o n s i s t e n t s t i m u l a t i o n c a u s e d by t h e p r e s e n c e o f r e s i d u e s t h r o u g h o u t t h e g r o w i n g p e r i o d . H o w e v e r , i t s h o u l d be p o i n t e d o u t t h a t , w h i l e t h e r a t e o f l e a f a r e a e x p a n s i o n may h a v e b e e n s t i m u l a t e d , t h e t o t a l l e a f a r e a s o f t h e p l a n t s grown i n t h e p r e s e n c e o f r e s i d u e s w e r e c o n s i s t e n t l y a n d s u b s t a n t i a l l y l e s s t h a n t h o s e o f t h e c o n t r o l s , as r e v e a l e d by F i g . 13 and 1 4 . The e a r l y p e a k s i n t h e f i t t e d c u r v e s f o r R, E, LAR, a n d 0 1 f o r r e s i d u e - g r o w n p l a n t s ( F i g . 12) a r e t h e r e f o r e t h e r e s u l t o f t h e d e l a y i n t h e o n s e t o f a p p r e c i a b l e g r o w t h . Thus d u r i n g t h e f i r s t 14 d a y s o f g r o w t h , t h e s e p l a n t s a c c u m u l a t e d l i t t l e d r y m a t t e r , s o t h a t i n r e l a t i v e t e r m s , t h e i r s l o w g r o w t h d u r i n g t h e s u b s e q u e n t 14 d a y s n e v e r t h e l e s s r e v e a l e d i t s e l f t h r o u g h r e l a t i v e g r o w t h r a t e s c o n s i d e r a b l y g r e a t e r t h a n t h o s e o f t h e c o n t r o l s . F i g u r e s 13 and 14 p r e s e n t t h e a c t u a l a n d f i t t e d d a t a f o r W, L and LAR and t h e f i t t e d c u r v e s f o r R f R L , E and a , b a s e d o n t h e p o l y n o m i a l e q u a t i o n s d e r i v e d f o r W a n d L ( A p p e n d i x 1 ) . No s t a t i s t i c a l c o m p a r i s o n c a n be made b e t w e e n t r e a t m e n t s s i n c e t h e f i t t e d c u r v e s w e r e b a s e d on mean v a l u e s o n l y . 50 F i g u r e 12. Comparison of growth parameters of mungbean grown on s o i l s w i t h r e s i d u e s ( • ) vs s o i l s without r e s i d u e s ( o ). U n i t s used a r e : -1 , -1 R — mg . g . day 2 -2 -1 R L — cm . dm . day -2 -1 E — mg . dm . day LAR — dm2 . g - 1 a ~ R/RL These are averages across i n c u b a t i o n p e r i o d s . 51 DAYS AFTER E M E R G E N C E F i g u r e 13. T o t a l dry weight (W), l e a f area (W L), r e l a t i v e growth r a t e (R), r e l a t i v e l e a f area growth r a t e (R^), net a s s i m i l a t i o n r a t e ( E ) , l e a f area r a t i o (LAR) and o of the succeeding mungbean crop grown on s o i l c o n t a i n i n g l e a f and r o o t r e s i d u e s a f t e r a one-week i n c u b a t i o n . U n i t s as f o r F i g u r e s 11 and 12. Fallow : • - a c t u a l data; o- f i t t e d data; s o l i d l i n e . Residue : • - a c t u a l data; °- f i t t e d data; dashed l i n e . D A Y S A F T E R E M I R G E N C E F i g u r e 14. T o t a l dry weight (W), l e a f area ( L ) , r e l a t i v e growth r a t e (R), net a s s i m i l a t i o n r a t e ( E ) , r e l a t i v e l e a f area growth r a t e ( R T ) , l e a f area r a t i o (LAR) and a of the succeeding mungbean crop grown on s o i l c o n t a i n i n g l e a f and r o o t r e s i d u e s a f t e r a three-week i n c u b a t i o n p e r i o d . U n i t s as f o r F i g u r e s 11 and 12. Fallow : A - a c t u a l data; A- f i t t e d data; s o l i d l i n e . Residue : •- a c t u a l data; f i t t e d d a t a ; dashed l i n e . 55 56 A comparison of Figures 13 and 14 reveals that the two sets of control plants behaved s i m i l a r l y . Thus they demonstrated s i m i l a r o v e r a l l growth curves for dry weight and leaf area i n terms of both form and magnitude. The same i s generally true for the derived growth parameters, although there are greater differences revealed with respect to the magnitude of some of the values. This i s probably the r e s u l t of s l i g h t differences i n the growing conditions to which the plants were subjected, because of the two-week difference i n time of seeding. The only parameter showing a markedly d i f f e r e n t trend over time i s a . However, in both sets, the o v e r a l l trend i s for a to decline s l i g h t l y from a value close to 2, i . e . the demonstration of a s h i f t from a quadratic towards a l i n e a r r elationship between W and L. Inspection of Figures 13 and 14 also shows that the enhancement of the derived growth parameters within the early stages of growth caused by residues was greater following 3-week incubation. Effects of leaf and root residues. With the e f f e c t of combined leaf and root residues amply demonstrated i n Experiment 3a, i t became of i n t e r e s t to f i n d out which of the two residues was the source of the greater t o x i c i t y . With this information, investigation 57 of the mechanics of phototoxicity and the i n d e n t i f i c a t i o n of the phytotoxic compounds responsible could be focussed on a s p e c i f i c source. Experiment 3b was designed to compare the separate effects of le a f residues with those of root residues. The experiment was generally the same as Experiment 3a with the addition of treatments i n which only leaf or root residues were incorporated into the s o i l . Because of an early problem of establishing the plants, which was probably due to using too cold water for watering the pots at the s t a r t , some pots had to be discarded. As a r e s u l t only three samplings were possible, i . e . , 30, 52, and 75 days after emergence. As with Experiment 3a, t o t a l dry weights, component weights (leaves, stems, roots and pods), l e a f areas, LAR's, LWR's and SLR's were subjected to analyses of variance. Comparisons and contrasts of treatment e f f e c t s were done i n the following manner: a) with vs without leaf residues K1 = 1/2(T x + T 3) vs x 2 = 1/2(T 4 + T 2 ) ; b) with vs without root residues x 3 = 1/2 (T 2 + T 3) vs x 4 = 1/2 (T 4 + T^) ; where T^ = mean of le a f residue treatment; T 2 = mean of root residue treatment; T^ = mean of leaf and root residue treatment; T. = mean of control 58 The e f f e c t s of the mixed l e a f and r o o t r e s i d u e s on growth which had been observed i n Experiment 3a were again demonstrated and were shown to be more pro-nounced i n the case of the l e a f r e s i d u e treatments than the r o o t r e s i d u e treatments. Table 8 summarizes the c o n t r a s t between the treatments w i t h o r without l e a f r e s i d u e s and treatments with o r without r o o t r e s i d u e s on the components o f growth and t o t a l dry weight. I t can be c l e a r l y seen t h a t the h i g h l y s i g n i f i c a n t e f f e c t s of r e s i d u e s a l r e a d y presented i n Table 6 were mainly due to the presence o f l e a f r e s i d u e s . The d i f f e r e n c e s i n the e f f e c t s o f the two types of r e s i d u e s on the growth o f a succeeding mungbean crop are g r a p h i c a l l y i l l u s t r a t e d i n F i g u r e s 15 and 16. The primary data are presented i n Appendix 5. The magnitude of the e f f e c t s of the presence o f l e a f r e s i d u e s on l e a f area and the components of dry weight are c l e a r l y shown i n F i g . 15. In each case the presence o f l e a f r e s i d u e s r e s u l t s i n a decrease i n l e a f area or weight a t each h a r v e s t . In r e l a t i v e terms, the g r e a t e s t r e d u c t i o n i s i n the weight o f r o o t s . However, i t should a l s o be noted t h a t , because o f the r e l a t i v e l y g r e a t e r e f f e c t on t o t a l dry weight than on l e a f a r e a , o r l e a f weight, both LAR and LWR are i n c r e a s e d s l i g h t l y but s i g n i f i c a n t l y . With regard to the e f f e c t s o f r o o t r e s i d u e s d e p i c t e d i n F i g . 16, the d i f f e r e n c e s a t a l l h a r v e s t dates are reduced t o n o n - s i g n i f i c a n c e i n r e l a t i o n to the e f f e c t s 59 T a b l e 8. Summary o f c o n t r a s t s b e t w e e n t r e a t m e n t s w i t h v s w i t h o u t l e a f r e s i d u e a nd t r e a t m e n t s w i t h v s w i t h o u t r o o t r e s i d u e . w i t h v s w i t h v s l e a f x w i t h o u t w i t h o u t r o o t V a r i a b l e s L e a f r e s i d u e R o o t r e s i d u e I n t e r a c t i o n T o t a l d r y w e i g h t L e a f a r e a L e a f w e i g h t Stem w e i g h t R o o t w e i g h t P o d w e i g h t LAR LWR SLA ** — S t a t i s t i c a l l y s i g n i f i c a n t a t 1% l e v e l . * — S t a t i s t i c a l l y s i g n i f i c a n t a t 5% l e v e l , n s — N o n - s i g n i f i c a n t ** ** ** ** ** ** ** ** ns * * * ns ** ns ns n s ns ns * ** n s * ** ns dm ^/g/pot 4|— . SLA d r r 2 /g /po t 3 r 01-LAR 3 0 5 2 7 5 0 3 0 52 DAYS' A F T E R E M E R G E N C E Figure 15. The e f f e c t o f l e a f residue on the t o t a l dry weight (W), l e a f area ( L ) , l e a f (W ), stem (W ), ro o t (WR) weights, l e a f weight ratio(LWR), s p e c i f i c l e a f area r a t i o (SLA) and l e a f area r a t i o (LAR). A - without l e a f r e s i d u e ; A - w i t h l e a f r e s idue. Regression l i n e s f o r W, L, W_ and W„ are h i g h l y s i g n i f i c a n t ; the r e s t are n o n - s i g n i r i c a n t . 61 g/pot 15 10 o dm. /po t 15 0l L OL g/pot 5 g/pot 4 r OL g/pot OL d m 2 /g/pof 4 r 01-30 _ L g/g/pot 0 dm^/g/p-ot 3r-OL 52 75 D A Y S A P T E R L W R 30 E M E R G E N C E L A R 52 "A 75 Figure 16. The e f f e c t of root residue on the t o t a l dry weight (W), l e a f area ( L ) , l e a f (WO, stem (W_), root (W ) weights, l e a f weight r a t i o (LWR), s p e c i f i c l e a f area r a t i o (SLA), and l e a f area r a t i o (LAR). A -without r o o t residue; • -with root residue. The above r e g r e s s i o n l i n e s are n o n - s i g n i f i c a n t . 62 of l e a f r e s i d u e s . The e x c e p t i o n i s r o o t weight which i s again s i g n i f i c a n t l y reduced. I n s p e c t i o n of Table 8 a l s o shows there were s e v e r a l s i g n i f i c a n t i n t e r a c t i o n s between the e f f e c t s o f l e a f and r o o t r e s i d u e s , the most s i g n i f i c a n t b e i n g those on r o o t weight and LWR. To make the nature and q u a n t i t a t i v e value o f the i n t e r a c t i o n s between l e a f and r o o t r e s i d u e s more apparent, use has been made o f the di a g r a m a t i c r e p r e s e n t a t i o n d e v i s e d by Richards (1941). In such diagrams, the a b s c i s s a e r e p r e s e n t s u c c e s s i v e increments i n the l e v e l o f one f a c t o r or the i n t r o d u c t i o n o f a d d i t i o n a l f a c t o r s . The co o r d i n a t e s r e p r e s e n t the magnitude of the e f f e c t on the v a r i a b l e i n q u e s t i o n . For example, i n t h i s experiment the a b s c i s s a e f o r R ichards' diagrams f o r treatment i n t e r a c t i o n s run from zero t o 2: L e v e l Leaf r e s i d u e L e v e l 0 1 Root 0 (Treat. 4) (Treat. 1) Residue 1 (Treat. 2) (Treat. 3) Treatment 4 corresponds t o "CV1, treatments 1 and 2 to "1" and treatment 3 to " 2 " , ( a b s c i s s a e v a l u e s ) . 63 Thus, a and b below are two possible configurations that may r e s u l t from a 2 x 2 f a c t o r i a l experiment: In these examples, s o l i d l i n e s reveal the e f f e c t s of l e a f residues, and broken l i n e s those of root residues. The above diagrams i l l u s t r a t e the form and magnitude of in t e r a c t i o n s . Where there are no interactions between two factors, the diagram takes the form of a parallelogram, with each factor acting independently. The absence of p a r a l l e l i s m indicates the i n t e r a c t i o n between two factors. F i g . 17 shows the Richards' diagram fo r the leaf and root interactions indicated i n Table 8. I t w i l l be noted immediately that there i s an absence of p a r a l l e l i s m i n most of the diagrams which indicates that there are interactions i n the e f f e c t s of both types of residues on the components of growth, although not a l l reach s t a t i s t i c a l s i g n i f i c a n c e (Table 8), e.g. Wp and L. The highly 64 q/poi 0 - 9 T 4 0-6 0-3 0»-\ 2 g/pot 3 T > ^-3 0 dm /g/pot 2r-01 L 2 LAR _L g/g/pof 0-5 O U 4— LWR J L dm 2 /g/pot )i- 4.. 'S L A 0-L — L F i g u r e 17. Richard's ( f a c t o r ) diagram of r o o t x l e a f r e s i d u e i n t e r a c t i o n s . Means across sampling d a t e s . 1 - l e a f r e s i d u e 3 - r o o t l e a f r e s i d u e 2 - r o o t r e s i d u e 4 - c o n t r o l 65 s i g n i f i c a n t i n t e r a c t i o n s on r o o t weight and LWR show t h a t the presence of e i t h e r r e s i d u e alone r e s u l t s i n a c l o s e to maximal e f f e c t . On the o t h e r hand, the e f f e c t s on t o t a l weight, the weights of l e a v e s , stems and pods, and on l e a f area are c l e a r l y shown to be caused by l e a f r a t h e r than r o o t r e s i d u e s . The r e l a t i v e l y g r e a t e r e f f e c t of l e a f r e s i d u e on W than on L and WL i s c l e a r l y i l l u s t r a t e d i n the diagram i n which the values f o r LAR, LWR and SLA w i t h l e a f r e s i d u e are s l i g h t l y h i g h e r from those w i t h r o o t r e s i d u e . F i g u r e s 18 and 19 p r e s e n t f i t t e d curves f o r R, E, R and o computed from the polynomials d e r i v e d L from the means o f W and L (Appendix 1 ) . Although no s t a t i s t i c a l comparisons can be made, i t can be seen t h a t both r e s i d u e sources cause s i m i l a r trends i n the r e l a t i v e growth r a t e , r e l a t i v e area growth r a t e and a . In the case of net a s s i m i l a t i o n r a t e s , the e f f e c t of l e a f r e s i d u e s was to cause a r e d u c t i o n throughout the growth p e r i o d , w h i l e w i t h r o o t r e s i d u e s , the r a t e s were maintained throughout and e v e n t u a l l y exceeded those o f p l a n t s grown i n the absence o f r o o t r e s i d u e s . In g e n e r a l , the values o f o show an i n c r e a s e w i t h time, i n d i c a t i n g a t l e a s t a maintenance of the q u a d r a t i c r e l a t i o n s h i p between W and L. However, i t i s apparent t h a t the e a r l y s t i m u l a t i o n s o f R, E and R^ demonstrated i n Experiment 3a were not repeated i n Experiment 3b, p o s s i b l y because o f the d i f f e r e n c e s i n the season of the y e a r a t which the d i f f e r e n t experiments were conducted. 66 80 r 6 0 h 4 0 h 20h L E A F RESIDUE v R v • 80 • v 60 4 0 20 0*-R O O T R E S I D U E R v 50r 25 0*-30 v v 50 25 A T o I 1-v •- T V 52 75 30 52 DAYS A F T E R E M E R G E N C E T V 75 F i g u r e 18. The e f f e c t o f l e a f and r o o t r e s i d u e s on r e l a t i v e growth r a t e and net a s s i m i l a t i o n r a t e o f the second crop o f mungbeans. • - w i t h r e s i d u e ; v - without r e s i d u e . -1 -1 -2 -1 R: mg . g . day ; E: mg . dm . day 67 6 r L E A F R E S I D U E • v 0 1 - ' ¥ _J_ o 1-R O O T R E S I D U E v T • V 8 | 8 r A A A o 1 _L 30 X 52 7 5 3 0 5 2 D A Y S A F T E R E M E R G E N C E 75 F i g u r e 19. The e f f e c t o f l e a f and r o o t r e s i d u e s on the r e l a t i v e l e a f a r e a growth r a t e and R/R^ r a t i o o f the second crop o f mungbean. • - w i t h r e s i d u e ; v - without r e s i d u e . 2 -2 -1 R L: cm .dm . day ; a : R/Rj^ 68 DISCUSSION Residue e f f e c t s . The evidence i m p l i c a t i n g the leaves as the main source of p h y t o t o x i n causing the r e s i d u e problem i n s e q u e n t i a l mungbean cropping i s q u i e t c l e a r . The a bsolute values of t o t a l dry weight a t any sampling date o f p l a n t s growing i n s o i l w i t h l e a f and r o o t r e s i d u e s (Experiment 3a) i s about 50% of the c o n t r o l values i n the case o f the one-week i n c u b a t i o n p e r i o d and about 40% f o r the 3-week i n c u b a t i o n . Where the e f f e c t o f l e a f and r o o t r e s i d u e s are separated (Experiment 3b), t o t a l dry weight a t t a i n e d 43% and 75% o f c o n t r o l s f o r p l a n t s grown r e s p e c t i v e l y i n s o i l w i t h l e a f - and r o o t - r e s i d u e s . I t has been observed i n f i e l d experiments t h a t s o i l w i t h mungbean r e s i d u e s which are kept moist has the g r e a t e s t e f f e c t on the subsequent mungbean crop and t h a t d r y i n g of the s o i l p r o g r e s s i v e l y reduces the magnitude o f t h i s e f f e c t (Runeckles, 1975). Such o b s e r v a t i o n may account f o r the g r e a t e r e f f e c t o f the 3-week i n c u b a t i o n s i n c e i n t h i s study the growing medium was maintained i n a moist c o n d i t i o n (see M a t e r i a l s and Methods s e c t i o n ) . The g r e a t e r b u l k o f the l e a f dry matter over t h a t o f r o o t s , however, may not account f o r the g r e a t e r absolute magnitude of the l e a f r e s i d u e e f f e c t s . The f i r s t crop of Experiment 3b had a 3:1 l e a f - r o o t weight r a t i o 69 on the average. Table 9 presents the weight r e d u c t i o n i n p e r c e n t o f c o n t r o l due to l e a f , r o o t and l e a f p l u s r o o t r e s i d u e s . Table 9. Dry weight r e d u c t i o n of mungbean at f i n a l h a r v e s t , i n p e r c e n t o f c o n t r o l , caused by the i n c o r p o r a t i o n i n the s o i l o f l e a f , r o o t and r o o t p l u s l e a f r e s i d u e s o f p r e v i o u s mungbean crop. V a r i a b l e s l e a v e s ( L ) Residues roots(R) L + R T o x i c i t y L/R perc e n t T o t a l dry weight 53.7 13.8 52.6 3. 89 Component weights: leaves 43.5 13.2 46.7 3.29 stem 54.1 10.4 50.7 5.2 root 42.6 18.4 50.1 2.3 pods 61.2 15.0 57. 5 4.1 In t h i s experiment, were incubated f o r i t w i l l be twelve days r e c a l l e d . I t w i l l t h a t the r e s i d u e s be noted t h a t the l e a f - r o o t r e s i d u e mix caused a r e d u c t i o n o f 52.6% on t o t a l dry weight which c l o s e l y c o r r o b o r a t e s t h a t o f the r e d u c t i o n caused by one-week i n c u b a t i o n (52.3%) i n Exper-iment 3a. On a l a n d area b a s i s under f i e l d c o n d i t i o n s , t h e r e f o r e , there are more l e a f r e s i d u e s than r o o t r e s i d u e s from the pre v i o u s mungbean crop. However, assuming t h a t the p h y t o t o x i n i s evenly d i s t r i b u t e d i n the p l a n t t i s s u e s , i t i s apparent t h a t the l e a f r e s i d u e which i s shown to 70 cause about 4 times more r e d u c t i o n of t o t a l dry weight (Table 9) i s 12.3% (53.7 - 13.8 x 3 ) more t o x i c on a p r o p o r t i o n a t e r e s i d u e weight b a s i s . Leaf r e s i d u e caused even more r e d u c t i o n of stem and pod weight, 22.9% and 16.2% r e s p e c t i v e l y . As to why the l e a c h a t e s of decomposing leaves (Experiment 2) d i d not show p h y t o t o x i c i t y can o n l y be surmised here. I t c o u l d p o s s i b l y be understood once the i d e n t i t y and nature of the p h y t o t o x i n i s known. There are some pot e n t i n h i b i t o r s , such as aglycones, which are o n l y very s l i g h t l y s o l u b l e i n water (Rice, 1974). I t i s a l s o p o s s i b l e t h a t the harmful e f f e c t s of mungbean r e s i d u e s behave s i m i l a r l y t o the f i n d i n g s of P a t r i c k e t a l . (1963) i n t h a t r o o t i n j u r y t o l e t t u c e and spinach s e e d l i n g s was c o n f i n e d mainly to those p a r t s i n d i r e c t c o n t a c t w i t h or i n the immediate v i c i n i t y of decomposing p l a n t fragments i n the s o i l . Organisms i s o l a t e d from l e s i o n s at the p o i n t o f i n j u r y were found to be mostly nonpathogenic and p h y t o t o x i c substances were presumed to have been e x t r a c t e d from p l a n t r e s i d u e s t h a t had decomposed under n a t u r a l c o n d i t i o n s f o r v a r i o u s p e r i o d s . The presence of some p h y t o t o x i c i t y from the r o o t s (Experiment 3b), on the other hand, can be assumed to be a t t r i b u t a b l e to some breakdown products o f the r o o t t i s s u e s s i n c e l e a c h a t e s from the r h i z o s p h e r e o f i n t a c t p l a n t s (Experiment 1) were not p h y t o t o x i c . T h i s may be s i m i l a r to the case o f A f r i c a n m a r i g o l d CTagetes erecta), 71 which contains a c t i v e nematocides i n i t s r o o t s , but which has f a i l e d to y i e l d i s o l a t e s of the compounds from the exudates of i n t a c t r o o t s (Clayton and Lamberton, 1964). The p o s s i b i l i t y cannot be r u l e d out, however, t h a t the r o o t exudates may c o n t a i n compounds which when degraded by microorganisms produce p h y t o t o x i n s and t h a t the e xperimental c o n d i t i o n s were not f a v o r a b l e f o r m i c r o b i a l growth. Since no b i o a s s a y was done on l e a f t i s s u e e x t r a c t s , i t i s not p o s s i b l e to t e l l whether the phyto-t o x i n i s p r e s e n t i n the l e a f t i s s u e s o r i s only formed and r e l e a s e d by decay. There i s always the p o s s i b i l i t y t h a t n o n - t o x i c compounds i n the p l a n t t i s s u e s may be transformed to t o x i c ones by m i c r o b i a l metabolism. A good example i s the case of amygdalin i n peach r o o t r e s i d u e ( P a t r i c k , 1955). Growth parameters. Except f o r R^, the d e r i v e d growth parameters of Experiment 3a show l i t t l e s i m i l a r i t y to those of Experiment 3b. Probably, growth was i n f l u e n c e d by season of p l a n t i n g . Experiment 3a, which was p l a n t e d i n summer (July 26, 1978), had the h i g h e s t dry matter accumulation. F i g u r e 20 presents the extent of r e d u c t i o n of dry matter accumulation due to season of p l a n t i n g 2 (r =0.769); the negative c o r r e l a t i o n (r= -0.877) i s s i g n i f i c a n t at the 5% l e v e l . As shown i n the f i g u r e , Experiments 3a and 3b were grown d u r i n g two d i s t i n c t seasons where the maximum p o s s i b l e l i g h t d u r a t i o n changed from 16 to 13.5 hours and from 10 to 8.2 hours r e s p e c t i v e l y . 72 g/pot 4 0 r ^ y = 3 6 - ( 2 9 3 4 5 - 0 - l 7 8 7 4 | f X r = 0 - 8 7 7 * 0 |_ y=22 0 8 - O I 2 9 7 9 3 8 X r= 0-7/04 ns - i — i — i — _ J I 1 !_ Jul 2 6 ' 7 8 9 ' 7 8 2 3 ' 7 7 Exper iments 3a 3a I 14 28 8 0 N U M B E R O F D A Y S A u g Aug Oct l 4 ' 7 7 A JLI l_ l.03: 110 Nov Nov 6 ' 7 8 I3'77 3b 2 Figure 20. Reduction i n dry weight of tops of mungbean as influenced by date of planting. Each observation represents an average of the control treatment. • : harvested at maturity (70-75 DAE) A : harvested at reproductive stage (25-35 DAE) 7 3 Light q u a l i t y and in t e n s i t y under the glass roof during these two periods would have been also d i f f e r e n t , the l a t e r season being more cloudy. Poehlman (1978) reported l i t t l e success with mungbean variety f i e l d t r i a l s grown above 40° l a t i t u d e , apparently due to the delay i n flowering re s u l t i n g from long photoperiod or poor growth due to the cooler temperature. Mean minimum temperature for productive growth appears to be between 20° and 2 2°C with the optimum mean temperature i n the range of 2 8°-30°C. In both Experiments 3a and 3b, temperature was within the above ranges (see Materials and Methods) and apparently, flowering was not affected by l i g h t duration since there was no observed change i n the days-to-flowering time. This observation suggests that the difference i n dry matter accumulation would have been due to the difference i n l i g h t i n t e n s i t y and spectral d i s t r i b u t i o n . This problem was not anticipated. I t was thought that the Lucalox Sodium-vapor lamps, which d e l i v e r l i g h t i n the photosynthetically active region of the l i g h t spectrum, were adequate. Although the growth pattern i n Experiment 3b was d i f f e r e n t from that of Experiment 3a, the reduction i n dry matter accumulation due to residue, which i n Experiment 3b was c l e a r l y shown to be due mainly to leaf residue, i s f a i t h f u l l y consistent. The derived growth parameters of the control treatments i n Experiment 3a however agree very closely to the ones reported by Tsiung (1978) . 74 There i s f u r t h e r s i m i l a r i t y between Experiments 3a and 3b. F i g u r e s 21 and 2 2 p r e s e n t the comparative e f f e c t s o f the r e s i d u e treatments i n two Experiments on the p a r t i t i o n i n g of a s s i m i l a t e s over time. These f i g u r e s summarize the data p r e s e n t e d i n Appendices 6 through 9. I t w i l l be noted t h a t the presence o f r e s i d u e s s t i m u l a t e d the accumulation o f more a s s i m i l a t e s i n the leaves a t the v e g e t a t i v e stage, i . e . , up to 2 8 days a f t e r emergence ( F i g . 21). T h i s s t i m u l a t i o n was more pronounced as the i n c u b a t i o n p e r i o d o f the r e s i d u e was brought up to 3 weeks. But the i n c r e a s e d a s s i m i l a t o r y s u r f a c e , however, d i d l i t t l e f o r the recovery o f the p l a n t , i n s p i t e o f the apparent s t i m u l a t i o n of E and R at t h a t p e r i o d ( F i g . 12) s i n c e the residue-grown p l a n t s a t t a i n e d only about 50% of the t o t a l dry weight of c o n t r o l a t f i n a l h a r v e s t . The same s t i m u l a t e d a l l o c a t i o n of a s s i m i l a t e s to the leaves i s shown wit h l e a f r e s i d u e s alone, and to a l e s s e r degree with r o o t r e s i d u e s alone, i n F i g u r e 22, which a l s o shows the delay i n pod formation among l e a f - r e s i d u e - t r e a t e d p l a n t s which was not observed i n Experiment 3a. T h i s delay i n pod formation may a l s o be due i n p a r t to s e a s o n a l e f f e c t s , as d i s c u s s e d e a r l i e r . The most s t r i k i n g o b s e r v a t i o n i n Experiment 3a i s the apparent s t i m u l a t o r y e f f e c t o f r e s i d u e on the d e r i v e d growth parameter E p a r t i c u l a r l y a t the v e g e t a t i v e stage ( F i g . 12). Since E r e p r e s e n t s the net p h o t o s y n t h e t i c g a i n over r e s p i r a t o r y l o s s and may vary a c c o r d i n g to the magnitude of r e s p i r a t i o n (Leopold and Kriedemann, 1975), °/ /o 14 28 42 73 14 28 42 73 DAYS AFTER EMERGENCE WITH R E S T D U E W I T H O U T R E S I D U E F i g u r e 21. Component dry weights as p e r c e n t of t o t a l dry weight o f succeeding mungbean crop as a f f e c t e d by the r e s i d u e and l e n g t h o f i n c u b a t i o n o f pre v i o u s mungbean crop. /o 3 0 52 75 30 52 DAYS AFTER E M E R G E N C E ROOT R E S I D U E ( R ) L + R ROOTS STEMS LEAVES PODS F i g u r e 2 2 . Component dry weights as p e r c e n t o f t o t a l dry weight o f succeeding mungbean crop as a f f e c t e d by the l e a f and/or r o o t r e s i d u e s o f previous mungbean crop. 7 7 the apparent greater magnitude of the rate of increase of R during the vegetative stage r e l a t i v e to the increase in assimilatory surface (Fig. 12) may account for the greater value f o r E over th i s period. This w i l l imply that there was less respiratory loss of assimilates during the period. This may suggest that the phytotoxin from the residue acts as a respiratory i n h i b i t o r to growing seedlings af t e r i t has caused i n i t i a l damage during the germination process. I t w i l l be r e c a l l e d that there was more assimilate allocated to the leaves among the residue-grown plants (Fig. 21), thus further inhancing increased net photosynthesis, over those plants grown without residues. This pattern of e f f e c t of stress i s s i m i l a r to that found of water-stressed tomato plants (Gates, 19 55) i n which, afte r w i l t i n g , lamina weight ratios became higher than those of controls, stem weight ratios became lower and E and R rose above control values. The present studies are i n keeping with the objectives of pinpointing the s p e c i f i c source of phytotoxin from the plant residue of previous crop and describing i t s e f f e c t on the growth parameters of the succeeding crop. The present studies do not permit any elaboration of the plant status e a r l i e r than 14 days after emergence. However, the implications of the o v e r a l l results point out that the e f f e c t starts right at the germination process. The status of the plant at the time of sampling (or observation) r e f l e c t s the cummulative consequence of whatever the plant was subjected to e a r l i e r . Hence, 7 8 the observed differences i n the growth parameters between plants grown i n residue-treated s o i l and those from residue-free s o i l suggest the need to elaborate the e f f e c t observed at the time of emergence as indicated by the conditions of the seedlings (Figs. 2 through 10) of the residue-treated s o i l . Obviously, i t would be inte r e s t i n g to determine the e f f e c t of leaf residue on the germination process, and p a r t i c u l a r l y i t s e f f e c t on r e s p i r a t i o n . Effects on early stage of growth, such as during elongation of the hypocotyl and tap root development, would also be i n t e r e s t i n g to know. Information along these l i n e s would be useful i n developing bioassay methods for i s o l a t e d compounds from leaf tissue extracts. Such information would also be useful i n developing screening procedures to survey the occurrence of the phytotoxin among mungbean c u l t i v a r s . 79 SUMMARY The purpose of this study was to investigate the effects of a mungbean crop on the growth parameters of a succeeding mungbean crop grown under various conditions of po t e n t i a l transfer and source of phytotoxicants. The mungbean variety, MG50-10a, used i n the experiments i s high-yielding and i s known to have residue problem i n a mungbean-mungbean sequential cropping. The residue problem appeared not to be a simple release of phytotoxin from root exudates of i n t a c t plants or of decaying plant materials. The e f f e c t i s more complex. The main results are summarized below: 1. Root exudates leached from growing plants i n sand medium did not show phytotoxicity. This suggests that root exudate per se i s non-phytotoxic. However, they may contain compounds which, through microbial metabolism, produce phytotoxins. 2. The residue e f f e c t was shown to be dependent on physical contact between subsequent crop roots and residues. Length of decomposition, up to 3 weeks, increased phytotoxicity. Leachate transferred from decomposing residues i n sand did not show phytotoxicity. Leaf residues were shown to be more phytotoxic than root residues. Leaf plus root residues were shown to have no additive e f f e c t . Residue treatment prevented normal seedling development and residue-grown plants attained about half the t o t a l dry weight of controls. Plants i n residue-treated s o i l have more assimilate allocated to the leaves during the vegetative stage, compared to those from residue-free s o i l . During this stage E, R, and LAR becomes considerably greater than for the controls. Although R L i s increased, which may be due to more assimilates being allocated to the leaves, the greater magnitude of the increase i n R over RT may account for the increase i n the value of E. This would be possible i f there i s a reduction i n respiratory losses, which suggests that the residues may be releasing a respiratory i n h i b i t o r . REFERENCES Anderson, R.C. and O.L. Loucks. 1966. Osmotic pressure influence i n germination test for a n t i b i o s i s . Science 152:771-772. B e l l , D.T. and D.E. Koeppe. 1972. Noncompetitive effects of giant f o x t a i l on the growth of corn. Agronomy J . 64:321-324. Borner, H. 1960. Liberation of organic substances from higher plants and t h e i r role i n the s o i l sickness problem. Bot. Rev. 26: 393-424. Buttery, B.R. 1969. Analysis of growth of soybeans as affected by plant population and f e r t i l i z e r . Can. J . Plant S c i . 49: 684-685. Chou, Chang-Hung and Z.A. Patrick. 1976. 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APPENDIX Polynomial equations f i t t e d to dry weights and l e a f areas and Tables of means of f i t t e d and a c t u a l data and other growth parameters of Experiments 3a and 3b. 87 APPENDIX 1 Polynomial equations f i t t e d to the mean t o t a l dry weight (W) and leaf area (L) data on sampling time ( t ) . Experiment 3a. Treat. 1 W 39941 - .18684t + .016089t 2 - .00013422t3 L 16887 + .14576t + ,007351t 2 - .000080782t3 Treat. 2 W 21569 + .020066t + .020216t2 - .0007209t3 L = — 50749 + •83727t - .0036837t2 - .000014149t3 Treat. 3 W • 62291 .27338t + .015732t 2 - .00013699t3 L 26105 - .041463t + .0058086t2 - ,000043845t Treat. 4 W *™ • 4742 -• • 389t + .039174t2 - .00037363t3 L =—te« 19406 + .27297t + .015845t2 - .00018043t3 3 Experiment 3b. Treat. 1 Treat. 2 Treat. 3 Treat. 4 W L W L W L W L .001 + .0012317t + .0012827t2 - .00000097851t3 .001 + .043475t + .0018639t2 - .000017437t3 .001 + .018902 + ,0012173t2 + .000010828t3 .001 + .10865t + .00085193t2 - .0000085666t3 .001 - .00087638t + ,0015323t2 - .000003547t3 .001 + .088115t + .00023259t2 - .0000051767t3 .001 + .063168t + .0063179t2 - .000037707t3 .001 + .10983t + .0024819t2 - .000027688t3 88 APPENDIX 2 Actual and f i t t e d data on the changes on mean t o t a l dry weights (W) i n Experiment 3a. Days After Actual F i t t e d Treatments - Emergence (g/pot of 4 plants) 1) One week 14 1.012 0.569 Incubation 28 4.254 4.835 42 11.270 10.989 73 20.260 20.284 2) Control for 14 3.748 3.987 Treat. 1 28 13.162 12.849 42 27.818 23.970 73 42.478 42.466 3) Three weeks 14 0.776 -0.497 Incubation 28 0.626 2.295 42 7.550 6.743 73 11.140 11.211 4) Control for 14 2.402 1.681 Treat. 3 28 11.148 12.093 42 26.016 25.558 73 35.452 35.487 89 APPENDIX 3 Actual and f i t t e d data on the changes on mean leaf areas (L) in Experiment 3a. Days After Actual F i t t e d Treatments Emergence (g/pot of 4 plants) 1) One week 14 2. 498 3.099 Incubation 28 8.698 7.910 42 12.562 12.943 73 18.260 18.227 2) Control for 14 8.566 10.453 Treat. 1 28 22.212 19.737 42 25.914 27.111 73 35.580 35.478 3) Three weeks 14 1.664 0.699 Incubation 28 1.426 2.692 42 6.130 5. 518 73 11.080 11.132 4) Control for 14 5.514 6.238 Treat. 3 28 16.860 15.911 42 25.394 25.854 73 34.018 33.980 APPENDIX 4 Means of t o t a l dry weight, l e a f area, dry weights o f l e a v e s , stems, r o o t s and pods. 2 (Weights are expressed i n g/pot o f 4 p l a n t s and l e a f area i n dm /pot of 4 p l a n t s ) . Experiment 3a. Days A f t e r Emergence V a r i a b l e s 14 28 42 73 1 week i n c u b a t i o n / c o n t r o l T o t a l dry weight 1. .01 3, .75 4, .25 13 .16 11. 27 23. 82 20. 26 42. 48 Leaf a r e a 2. .50 2, .57 8. .70 22 .21 12. 56 25. 91 18. 26 35. 58 Leaf dry weight 0. .60 1. .99 2. .27 6 .76 4. 29 8. 52 6. 98 12. 86 Stem dry weight 0. .27 1. .12 1. .50 4 .71 3. 10 6. 40 3. 82 8. 08 Root dry weight 0. .14 0. ,64 0. .48 1 .69 0. 98 2. 28 2. 10 3. 86 Pod dry weight - 2. 90 6. 62 7. 36 17. 68 3 weeks i n c u b a t i o n / c o n t r o l T o t a l dry weight 0. ,78 2. ,40 0. .63 11 .15 7. 55 26. 02 11. 14 35. 45 Leaf area 1. .66 5. ,51 1. .43 16 .86 6. 13 25. 39 11. 08 34. 02 Leaf dry weight 0. ,41 1. ,27 0. ,40 5 .56 2. 14 8. 08 3. 70 11. 84 Stem dry weight 0. .26 0. ,72 0. .13 4 .20 1. 78 7. 36 2. 10 9. 10 Root dry weight 0. ,12 0. ,41 0. ,09 1 .39 0. 49 2. 20 1. 12 3. 55 Pod dry weight — 3. 14 8. 38 4. 22 10. 96 91 APPENDIX 5 Means of t o t a l dry weights, l e a f area, dry weights of leaves, roots, and pods. (Weights are expressed i n g/pot of 4 plants and leaf area i n dm /pot of 4 plants). Experiment 3b. Treatments Variables Leaves Roots Root & Leaves Contn 30 DAE Total dry weight 1.166 1.956 1.258 2.774 Leaf area 2.212 3.797 2.713 4.781 Leaf weight .688 1.0 86 .708 1.378 Stem weight .380 .710 .460 .888 Root weight .098 .160 .090 .508 52 DAE Total dry weight 3.396 5.798 3.600 8. 498 Leaf area 4. 850 6. 751 4.563 8.532 Leaf weight 2.004 2. 878 2.038 3.520 Stem weight 1.118 1. 888 1.314 2.270 Root weight .274 .412 .248 1.282 Pod weight - .620 - 1.426 75 DAE Total dry weight 6.896 12.834 7.058 14.894 Leaf area 6.390 9. 327 5.732 10.518 Leaf weight 2. 202 3. 384 2.078 3.900 Stem weight 1. 596 3.118 1.714 3.480 Root weight .566 .804 .492 .986 Pod weight 2.532 5.548 2.744 6.528 9 2 APPENDIX 6 Root/Weight Rat i o s Experiment 3a Days A f t e r Emergence Treatment 14 28 42 73 1) 1 week i n c u b a t i o n .14 .11 .09 .10 2) C o n t r o l (1) .17 .13 .10 .09 3) 3 week i n c u b a t i o n .15 .14 .06 .10 4) C o n t r o l (3) .17 .12 .08 .10 Experiment 3b Days A f t e r Emerge nee Treatment 30 52 75 Leaf r e s i d u e (L) .08 .08 .08 Root r e s i d u e (R) .08 .07 .06 R & L .07 .07 .07 C o n t r o l .18 .15 .07 9 3 APPENDIX 7 Stem/Weight R a t i o s Experiment 3a Treatment 14 Days A f t e r 28 Emergence 42 73 1) 1 week i n c u b a t i o n .27 .35 .28 .19 2) C o n t r o l (1) .30 .36 .27 .19 3) 3 week i n c u b a t i o n .33 .21 .24 .19 4) C o n t r o l (3) .30 .38 .28 .26 Experiment 3b Days A f t e r Emergence Treatment 30 52 75 Leaf r e s i d u e (L) .33 .33 .23 Root r e s i d u e (R) .36 .33 .24 R & L .37 .37 .24 C o n t r o l .32 .27 .23 94 APPENDIX 8 Leaf/Weight R a t i o (W /W) Experiment 3a Days A f t e r Emergence Treatment 14 28 42 73 1) 1 week i n c u b a t i o n .59 .53 .34 .34 2) C o n t r o l (1) .53 .51 .36 .30 3) 3 week i n c u b a t i o n .52 .63 .28 . 33 4) C o n t r o l (2) .53 .50 .31 .33 Experiment 3b Days A f t e r Emergence Treatment 30 52 75 Leaf r e s i d u e (L) .59 .59 .32 Root r e s i d u e (R) .55 .50 .26 R & L .56 .57 .29 C o n t r o l .50 .41 .26 95 APPENDIX 9 a) Pod/whole p l a n t weight r a t i o . Experiment 3a Days A f t e r Emergence Treatment 42 73 1) 1 week i n c u b a t i o n .26 .36 2) C o n t r o l (1) .28 .42 3) 3 weeks i n c u b a t i o n .42 .38 4) C o n t r o l (3) .32 .31 b) Pod/whole p l a n t weight r a t i o Experiment 3b Days A f t e r Emergence Treatments 52 75 Leaf r e s i d u e (L) 0 .37 Root r e s i d u e (R) .11 .43 R & L 0 .39 C o n t r o l .17 .44 

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