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Presence and possible significance of the endophytic bacterial flora in solanum tuberosum l De Boer, Solke Harmen 1972

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THE PRESENCE AND POSSIBLE SIGNIFICANCE OF THE ENDOPHYTIC BACTERIAL FLORA IN SOLANUM TUBEROSUM L.  BY SOLKE HARMEN DE BOER B.Sc. (Agr.), University of B r i t i s h Columbia, 1970  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN THE DEPARTMENT OF PLANT SCIENCE  We accept this thesis as conforming to the required standard.  THE UNIVERSITY OF BRITISH COLUMBIA July, 1972  In p r e s e n t i n g t h i s t h e s i s  in p a r t i a l  f u l f i l m e n t o f the r e q u i r e m e n t s  an advanced degree at the U n i v e r s i t y o f B r i t i s h Columbia, the L i b r a r y s h a l l make i t  freely available  for  I agree  for  that  r e f e r e n c e and s t u d y .  I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s  thesis  f o r s c h o l a r l y purposes may be g r a n t e d by the Head o f my Department or by h i s r e p r e s e n t a t i v e s . of  It  this thesis for financial  written  gain shall  permission.  Department o f  Plant Science  The U n i v e r s i t y o f B r i t i s h Vancouver 8, Canada  Date  i s understood t h a t copying o r  July, 1972  Columbia  publication  not be a l l o w e d w i t h o u t my  ii ABSTRACT The t o t a l number of viable bacteria found i n Solanum tuberosum 3 7 stems and tubers was found to vary from less than 1 x 10 to k.J x 10 c e l l s - cm 3 i n tubers. About 75% per cm 3 i n stems and from 0 to 1.6 x 10 c e l l^s per of both stems and tubers had populations at the lower end of the range but there was no significant correlation between bacterial counts i n stems and tubers of the same plant.  Bacteria found i n potato tubers included species  of Micrococcus, Pseudomonas, Bacillus, Flavobacterium, Agrobacterium, and Xanthomonas. Also present were coryneforms and some others which were not identified to genus but were gram negative.  Some of the coryneforms were  morphologically indistinguishable from the bacterial ring rot organism (Corynebacterium sepedonicum) but non-pathogenic and biochemically different from i t . About 5% of stem smears of healthy plants showed more than 30 gram positive rods per microscope f i e l d . A l l the Bacillus spp., one Pseudomonas sp. and three unidentified species were found to inhibit C. sepedonicum i n v i t r o .  An antibiotic was  p a r t i a l l y purified from the Bacillus sp. showing the greatest amount of inhibition. pathogens:  This species was also antagonistic toward the following potato Pseudomonas solanacearum, Erwinia atroseptica, E. carotovora,  Alternaria solani, and Phytophthora infestans.  Physical and chemical tests  showed that both a l i p i d and a peptide antibiotic were involved i n the antagonistic effect.  iii  TABLE OF CONTENTS PAGE Part A. Endophytic Bacterial Flora i n Solanum tuberosum Literature Review  1  Introduction  1  Presence of Endophytic Bacteria  1  Mode of Entry  2  Significance as Latent Pathogenic Forms  3  Significance i n Bacterial Ring Rot Diagnosis  k  Effect of Virus Infection on Host Resistance  5  Object of Project  6  Materials and Methods  6  Plant Material  6  Quantitative Assay  7  Qualitative Assay  11  Results  1^  Quantitative Assay  1^  Qualitative Assay..  17  Discussion  23  Summary  26  Literature Cited  27  Part B.  The Inhibition of Potato Pathogens by an Endophytic Bacillus sp.  Literature Review  31  Introduction  31  Biological Control of Plant Diseases  31  Antibiotics i n Plant Pathology  33  Object of Project  35  Material and Methods  35  Isolation of Bacteria and Demonstration of Antagonism  35  Separation of the Active Substance  36  Determination of Properties of the Antibiotic  39  Results  ^0  Discussion  ^9  Summary  51  Literature Cited  52  y LIST OF TABLES PAGE Part A Table 1.  The t o t a l viable endophytic bacterial populations in stems and tubers of Solanum tuberosum  15  Table 2 . Generic i d e n t i f i c a t i o n of bacterial isolates from Solanum tuberosum Table 3 .  19  Some characteristics of coryneform bacteria isolated from Solanum tuberosum  21  Part B Table 1.  Amino acids detected i n the p a r t i a l l y purified antibiotic  kk  LIST OF FIGURES Part A Figure 1.  Agar droplet dilution plate  Figure 2.  Agar dilution droplet under dissection microscope  9 10  Figure 3- Frequency distributions of endophytic bacterial populations i n stems and tubers of Solanum tuberosum Figure  Frequency distribution of number of gram positive rods found i n stem smears  Figure 5-  16 18  Comparison of Corynebacterium sepedonicum with a nonpathogenic coryneform  22  Part B Figure 1.  Inhibition of several potato pathogens by an endophytic Bacillus sp  Figure 2.  ^1  Corynebacterium sepedonicum inhibited by the p a r t i a l l y purified antibiotic  ^3  Figure 3.  U.V. spectrumof p a r t i a l l y purified antibiotic  ^5  Figure k.  I. R. spectrum of p a r t i a l l y purified antibiotic  kG  Figure 5.  Inhibition of Corynebacterium sepedonicum by two components of the p a r t i a l l y purified antibiotic  ^7  vii ACKNOWLEDGMENTS  The author gratefully acknowledges the guidance and encouragement given during the research and writing of this thesis by Dr. R. J . Copeman. Gratitude i s extended to Dr. R. Stace-Smith, Canada Department of Agriculture, Research Station, Vancouver, who o r i g i n a l l y suggested the project and showed continual interest i n i t . Thanks are also due for their helpful suggestions to the other members of my graduate committee which included Dr. V. C. Runeckles, Department of Plant Science; Dr. N. S. Wright, Canada Department of Agriculture; Dr. T. H. Blackburn, Department of Microbiology; and Dr. E. E. Ishiguro, Department of Microbiology. Special thanks i s extended to Dr. N. S. Wright for supplying the potato tubers used i n this study and f a c i l i t i e s for PVS testing. Thanks also to Dr. J . Tremaine, Canada Department of Agriculture, Research Station, for doing the amino acid analysis; to Esther Lo and Beatrice Schroeder, also from the Canada Department of Agriculture, for operating the electron microscope; and to Randy Englar, Department of S o i l Science for doing the I.E. spectrum. Support for this work was from NEC operating grant A-62^0 and grants from the UBC Committee on Eesearch awarded to Dr. E. J . Copeman.  Part A Endophytic Bacterial Flora i n Solanum tuberosum  LITERATURE REVIEW INTRODUCTION At one time internal healthy plant tissue was thought to be sterile.  However, since Perotti (^5) found bacteria i n healthy root tissue,  this concept has changed.  I t i s generally accepted that plant tissue may  contain internal micro-organisms.  These organisms have been labelled  'endophytes' or 'endophytic bacteria' ( 5 8 ) . Endophytic bacteria are distinct from epiphytic bacteria which occur on the plant surface ( 3 6 ) . between endophytes and epiphytes may at times be obscure.  The distinction  For example, when  bacteria occur on leaf surfaces and i n the substomatal chambers  the  distinction i s not sharp. However, bacteria occuring i n the vascular systems of a stem ( 2 2 ) , are definitely endophytes.  Presence of endophytic bacteria The presence of non-pathogenic bacteria i n apparently healthy stem and tuber tissue of Solanum tuberosum has previously been investigated (22, 3 8 , 5 0 , 5 6 ) .  Several genera were found to be present as determined by  different biochemical reactions.  Bacterium radiobacter ( 5 0 ) , Aerobacter  cloacae and Bacillus megaterium (22) were identified among the species present. Bacteria have also been obtained from potato seeds and seedlings, although their occurrence was not common ( 2 2 ) .  2  H o l l i s (22) found the ratio of bacteria i n xylera and phloem to be 2 1 : 1 i n potato. He hypothesized that the bacteria are located i n vessels and sieve tubes. However, Lutman and Wheeler (38) from histological observations suggested that they occur i n the middle lamellae between.cell walls. Endophytic bacteria have also been reported i n other crops. Approximately  h0°/o  of unblemished tomato f r u i t s were found to contain internal  micro-organisms (^9)-  The genera encountered i n the f r u i t s included;  Xanthomonas, Pseudomonas, Aerobacter, Escherichia, Micrococcus, Flavobacterium, and Corynebacterium. per ml of f r u i t juice.  The number of organisms varied up to 10,000  Samish and Dimant C+8) have shown that fresh, healthy  cucumbers contain an endophytic bacterial population. 4,000 bacterial c e l l s per ml of cucumber juice. identified.  They found an average of  These micro-organisms were not  Bacteria have also been reported i n the stems of Phaseolus vulgaris,  and i n the tap roots of Medieago and Melilotus spp. ( 5 0 ) . In addition bacteria have been isolated consistently from storage organs of kohlrabi, red beet, turnip, sweet potato, and carrots ( 5 6 ) . The presence of endophytic bacteria in carrots has been recognized as a problem i n prolonged physiological experiments ( 2 5 ) .  Mode of Entry The manner by which the endophytic bacteria enter the plant tissues i s s t i l l largely one of speculation.  In those cases where bacteria are  isolated from the f r u i t , flowers are a probable port of entry (39)• This i s substantiated by the recovery of greater numbers of bacteria from the blossom end as compared to the stem end i n cucumbers (48). Moreover flowers have also  3  been known to act as a port of entry for some phytopathogenic bacteria e.g. Erwinia amylovora. Bacteria could enter the flower either through nectarthodes or along with pollen tubes. Entry into roots and storage tissue probably occurs from the soil.  Bacteria could enter through l e n t i c e l s and wounds as has been  shown for pathogenic organisms (53)? or by direct penetration into the undifferentiated meristematic tissue as was hypothesized by H o l l i s ( 2 2 ) . Once i n the root system the organisms could move throughout the plant. An attempt has been made to test the root entry hypothesis.  Potato  seedlings i n sand culture were subjected to heavy suspensions of Pseudomonas tabaci. Subsequent attempts to isolate P_. tabaci from the potato plants met with only limited success ( 2 2 ) . On vegetatively propagated crops bacteria may be transferred from year to year i n the plant tissue as i s Erwinia atroseptica, E. carotovora (21, MO  and Corynebacterium sepedonicum (58) i n potato.  Seeds  may also act as carriers but probably are less effective than vegetative organs ( 2 2 ) .  Significance as Latent Pathogenic Forms Very l i t t l e work has been done on what significance endophytic micro-organisms may have i n the host plant. Some bacteria are able to multiply i n non-host plants (2k); thus their presence may have a greater effect on the host plant when their population increases. Erwinia atroseptica and E. carotovora have been isolated from apparently healthy potato stems (MO.  Kennedy (33) has demonstrated that  h  Pseudomonas glycinea i s present internally i n field-grown soybean plants and seeds.  Cameron (9) reported the presence of Pseudomonas syringae i n  healthy cherry trees. K e i l and van der Zwet ( 3 2 ) , moreover, isolated Erwinia amylovora from symptomless pear and apple tissue.  In all these examples  pathogenic species exist as latent forms or subclinical infections.  Thus  pathogenic forms may be part of the endophytic f l o r a .  Significance i n bacterial ring rot diagnosis Diagnosis of bacterial ring rot of potato caused by Corynebacteriumsepedonicum (Spieck. and Kotth.) Skapt. and Burkh. i s based on the occurrence of a characteristic rot i n vascular bundles of stems and/or tubers.  Confirma-  tion depends on finding large numbers of gram positive bacteria i n smears from affected tissues ( 2 0 ) .  S t r i c t control measures are applied i n most areas  because severe losses may result from infection.  In B r i t i s h Columbia, for  example, l e g i s l a t i o n provides disposal and cleanup procedures for l o c a l and imported l o t s i n which the disease i s found (Domestic Bacterial Ring Rot Act (8).  Throughout Canada there i s a zero tolerance for the disease i n c e r t i f i e d  seed (Destructive Insect and Pest A c t , ( l 0 ) . To avoid use of infected tubers for seed, gram-stained stem smears from selected plants are examined ( 3 ) .  I f short, gram positive rods are  found the plants are considered suspect. This scrutiny of "healthy" plants i s based on the assumption that sub-clinical infection by _C. sepedonicum may carry the organism from year to year i n tubers. Walker (58) states that " i t i s possible for a tuber to carry the germs internally and produce symptomless plants and symptomless tubers although a l l three stages are infected."  5  However, no experimental evidence i s cited.  It i s moreover assumed that  other short gram positive rods are absent from potato stems, and that the endophytic f l o r a i n the stem reflects that i n the tuber.  Effect of virus infection on host resistance The effect of virus infection on the subsequent susceptibility to other diseases has been well documented. Geranium plants infected with any of four latent viruses are more susceptible to bacterial stem rot caused by Xanthomonas pelargoni ( 3 5 ) .  S i m i l a r i l y pea mosaic, a l f a l f a mosaic, bean  yellow mosaic, and pea enation mosaic virus increase susceptibility to pea root rots caused by Aphanomyces euteiches or Fusarium solani ( l 8 ) .  Sugar  beets infected with beet mild yellowing virus are more susceptible to Alternaria sp. and Erysiphe betae (V7)»  However, virus infection does not  always render the plant more susceptible to subsequent infection.  Suscepti-  b i l i t y was reduced to Cladosporium cucumerinum infection by the cucumber mosaic virus (23), and chlamydospore germination of Fusarium solani f. sp. cucurbitae was decreased by squash mosaic, water melon mosaic and wild cucumber mosaic viruses ( 1 7 ) .  These are only a few examples from a large  number of virus-fungus or virus-bacterium, interactions reported. Similar interactions i n potato involving latent viruses and pathogenic fungi and bacteria have been reported.  Potato plants infected with  potato virus X were more resistant to subsequent infection with late blight (Phytophthora infestans) ( 4 l ) , and early blight (Alternaria solani) (42). Potato virus Y induced similar resistance to late blight (kl) but decreased resistance to Alternaria solani (^2). Jones et a l (28) suggested that virus X  6  infected potatoes were more susceptible to Fusarium tuber rot. Ayers and MacKinnon (personal communication) f a i l e d to confirm this and their work indicated that X - free potatoes were more resistant to tuber rot. A v a r i e t a l difference i n response was found, however, and may account for the conflicting reports.  A synergistic effect was observed on the potato  plant when ring rot bacteria were inoculated into material infected with leaf r o l l virus (4-3). Since virus infection has an effect on potato diseases i t seems reasonable that i t may also have an effect on the endophytic bacterial f l o r a .  Object of Project This study was undertaken ( i ) to determine the bacterial population in Solanum tuberosum, ( i i ) to evaluate the effect of virus infection on this population, and ( i i i ) to identify the coryneform bacteria found i n healthy plants and compare them to C. sepedonicum.  MATERIALS AND METHODS Plant Material Two l o t s of Solanum tuberosum L. cv "Netted Gem" from the same parental clone were obtained from N. S. Wright (CDA, Vancouver).  One l o t  was virus-free (VF), the other had been infected with potato virus X (PVX). Plants were grown i n adjacent plots on the UBC campus.  In late summer the  plants were twice sprayed with Thiodan 4E for aphid control. In the f a l l stems and tubers were individually harvested and numbered. Stems were used immediately while the tubers were stored at 5 C for three weeks.  7 A l l plants i n the virus-free plot were indexed for PVX and PVS to ensure that they had not been contaminated during the growing season. The PVX test was done by taking a small piece of tissue from each plant and grinding i t with a few drops of water on a spot plate.  The juice was  then rubbed on Gomphrena globosa leaves with a square of foam rubber. The Gomphrena globosa plants had been trimmed to four leaves and dusted with carborundum (600 mesh Crystalon) prior to inoculation.  Local lesions on  the indicator plant denoted the presence of PVX. PVS was indexed by tube p r e c i p i t i n serology.  From each plant  0.25 gm of leaf tissue was ground i n a hand tissue grinder with 2 ml Tris (0.01 M) buffered 0 . 8 5 % saline at pH 7.4. The homogenate was centrifuged at 7OOOg_ for 20 minutes. Two s e r i a l two-fold dilutions were made of the 5  supernatant and 1 ml suitably diluted PVS antiserum was added to 1 ml of each dilution.  The tubes were incubated for 2 hrs at 37 C i n a water bath.  Floculation, which indicates a positive test, was observed under transmitted light.  One plant, found to be contaminated with PVS, was discarded.  Quantitative Assay Stems were washed i n a 10% solution of commercial bleach, rinsed and a i r dried. (3).  Stem smears were made by the method of Baribeau and Marcotte  The stems were cut with a scalpel and smeared on a slide along the  complete width.  Four smears were made per slide.  Reed's modification of  the gram stain was used (46). Slides were observed under o i l immersion at 1250x and gram positive bacteria counted per microscope f i e l d . chosen at random i n areas where bacteria were present. f i e l d s i n each smear was recorded.  Fields were  The mean of two  8  For estimating the number of viable bacterial c e l l s i n the stem, 5 mm sections taken at the s o i l l i n e were used.  After surface s t e r i l i z i n g  in a 10% solution of commercial bleach and rinsing 3 times i n s t e r i l e d i s t i l l e d water (SDW), the stem sections were ground with 5 ml of SDW i n a V i r t i s Model k3 homogenizer with the micro assembly at 32,000 rpm for 5 minutes.  Volumes were brought up to 10 ml and s e r i a l dilutions made by the  agar droplet method ( 5 1 ) . Pasteur pipette.  The agar droplets were made from k drops of a  In this manner a drop of 0.097 ml was obtained which closely  approximated 0.1 ml.  Thus 0.1 ml of the homogenate was transferred to a  molten agar tube containing 9-9 ml of media "NM" (30) i n a ^5 C water bath. Four s e r i a l dilutions were similarly made. Four replications of each dilution were placed on a petri dish- (Fig. l ) . ;  hrs.  Plates were incubated at 25C for h8  Microcolonies were counted under a dissecting microscope at 12 X  magnification.  Colony counting was f a c i l i t a t e d by drawing a grid on a cover  glass and placing i t over the droplet to be counted (Fig. 2 ) . Tuber samples were obtained after surface s t e r i l i z i n g the tuber i n 10% commercial bleach and rinsing 3 times i n SDW.  Plugs of tissue were  extracted from the vascular ring at the stem end with a modified cork borer. The cork borer had a bolt which could be screwed i n from the handle end to extrude the tuber plug.  A:specific number of turns extruded a measured  length of plug which was subsequently sliced off with a s t e r i l e scalpel. 'The diameter and thickness of the discs so obtained were 10 mm and 3 mm respectively. stem tissue.  They were homogenized and diluted i n the same manner as the  Fig. 1 An agar droplet dilution plate showing four replications of four dilutions. 10 , 2  From l e f t to right the dilutions are  l o \ 1 0 , and 1 0 . 6  8  Fig. 2 An agar dilution droplet under the dissecting microscope.  Note the grid on a glass coverslip super-  imposed upon the droplet to f a c i l i t a t e counting.  11 A l l procedures were carried out i n a laminar a i r flow bench. To test for contamination, a control was run for every 9 samples.  Controls  consisted of s t e r i l e s o l i d i f i e d pieces of 3% agar put through the same steps as the potato tissue.  Only one of 20 controls contained two bacterial  colonies. Stem and tuber sections of equal size adjacent to the homogenized samples were taken for fresh and dry weight determinations.  Dry weight was  determined by drying to constant weight at 80 C. Diameter was also measured for the stem sections. The bacterial numbers were calculated on a per volume basis. Analysis of variance was used to compare bacterial numbers i n virus-free with virus-infected plants.  The simple regression of bacterial numbers i n stems  on that i n corresponding tubers was calculated.  Both simple and multiple  regression analyses were done on bacterial numbers using fresh and dry weights as independent variables.  Sequential position i n the rows was also used as an  independent variable i n the multiple regression because s o i l conditions varied along a row.  Qualitative Assay In preliminary work pieces of potato tuber tissue were placed i n M523 broth ( 2 9 ) . After 2h hrs incubation, aliquots from the broth were streaked on to plates of the same media. Dissimilar colonies were isolated and retained for identification i n pure culture on nutrient agar slants at 5 C.  12  In subsequent experiments only gram positive rods were retained. Potato stem and tuber tissue were surface s t e r i l i z e d and ground up as i n the quantitative assay. The homogenate was streaked on to a medium which allowed rapid growth of Corynebacterium (5*01 a medium which was somewhat selective for s o i l Arthrobacter spp. (4-0), and a nutrient agar with sodium dichromate ( l ; 20,000) to inhibit gram negative bacteria (.^k).  Dissimilar  colonies were isolated, stained and gram positive isolates retained for further study. The following tests were performed for the identification of bacteria. The method described by Bradbury (6) was used with the  Gram stain.  exception that 0 . 5 % aqueous safranin was substituted for basic fuchsin. Acid FastStain.  The Ziehl-Neelson carbolfuchsin stain (19) was used  to detect acid fastness. Methylene blue was used as the counter stain. Motility . (52)  M o t i l i t y was determined by both the hanging drop technique  and with Adler's semi-solid media ( l ) . Flagellation.  Both electron microscope (EM) and light microscope  techniques were used to determine number and location of f l a g e l l a .  Forty-  eight hr cultures i n nutrient broth were diluted 1:1 i n SDW and the suspensions dropped on to grids previously coated with colodion and carbon films.  The grids were stained with 20% phosphotungstic acid at pH 7-2, a i r  dried and observed i n a P h i l l i p s 200 microscope.  The electron microscope  was operated by technicians at the CDA Research Station, Vancouver. For the light microscope the s i l v e r impregnation stain (5) was used. Spore test.  The presence of spores was determined by heating a 2 ml  aqueous c e l l suspension to 80 C for 15 min ( 6 ) .  V i a b i l i t y of the heated  suspension indicated the presence of spores.  In a l l cases the conclusion  was confirmed by observation using phase contrast light microscopy. Pigment production.  King's B medium (3*0 was used to test for the  production of fluorescent pigment i n Pseudomonas spp. Carbohydrate metabolism.  Peptone water containing 2 ml/l of a 1.6%  alcoholic solution of bromocresol purple with the appropiate  carbohydrate  at a f i n a l concentration of 1% was used to detect acid production ( 5 2 ) . Both glucose and lactose were used aerobically and anaerobically. Gas release was detected i n the aerobic tubes by the insertion of a Durham tube.  Anaerobic conditions were obtained by the addition of 0 . 3 % agar  (to prevent convection currents) and layering 1 cm s t e r i l e mineral o i l on top of the medium after inoculation. Cellulose digestion. Cultures were grown i n peptone water containing a s t r i p of Whatman #1 f i l t e r paper.  Maceration of the f i l t e r paper indicated  cellulose digestion ( 5 2 ) . Cultures were incubated for 3 weeks at 25 C. Indole production.  The presence of indole was tested by shaking a  4-day-old culture grown i n peptone water with 0.5 ml xylene.  A few drops  of Ehrlich's reagent was added and a pink color recorded as positive ( 1 2 ) . Gelatin hydrolysis.  Cultures were grown on plates containing nutrient  agar with 20% gelatin (W/V). Gelatin hydrolysis was observed after flooding the plate with a saturated ammonium sulfate solution.  Formation of haloes  around the colonies indicated a positive test ( 1 2 ) . Nitrate reduction.  Cultures to be tested were grown for 3 days i n  peptone water with 0 . 1 % KNO^.  The s u l f a n i l i c acid o<. _ naphthylamine spot  test method was used to test for the presence of n i t r i t e ( 5 2 ) . Further  Ik  reduction of nitrate was detected by the addition of a small amount (approx. 0.5 - 1 nig) of zinc dust which reduces nitrate to nitrate ( 1 2 ) . Pathogenicity tests.  A l l isolates obtained were tested for pathogenicity  in both potato and tomato. c e l l suspensions (1 x 10  Stem inoculations were made by injecting aqueous cells/ml) with a hypodermic syringe.  Pathogenicity  was indicated by chlorosis and marginal necrosis of l e a f l e t s and wilting of stems with the presence of a creamy exudate i n the vasular tissue.  RESULTS Quantitative Assay Preliminary experiments suggested that there may be a difference between the endophytic bacterial population of VF and PVX plants. k2 VF and k^> PVX plants were analyzed.  Subsequently  Although the mean bacterial numbers  (Table l ) were s l i g h t l y higher for the VE plants, the difference was not significant at the 5% l e v e l . Since no difference was found between VF and PVX plants the results were combined for the frequency distributions.  As shown i n Figure 3, the  tubers were categorized into ten groups on the basis of the bacterial population k 3 which ranged from 0 to 1.6 x 10 c e l l s per cm . Similarly, stems were placed in categories indicative of bacterial populations from less than 1 x 10^ to 7  3  k.7 x 10 c e l l s per cm . Most stems and tubers had bacterial populations toward the lower end of the scale but 25% contained more than 1.2 x 10 c e l l s  per cm 3 i n stems and 50 c e l l s per cm 3 i n tubers.  There was no significant correlation between bacterial numbers i n the stem with the corresponding tuber of the same plant.  There was also no  15  Table 1.  The viable endophytic bacterial population* i n stems and tubers of Solanum tuberosum  STEM  TUBER  VF Mean  PVX  4.1 x 10  Standard Deviation  1.3 x 1 0  2.6 x  8.0 x 10  5.1 x 10  Median  VF  9.7 x 7  5 10  1.4 x 1 0  7  + Based on 45 replications.  10  3  6.6 9.4 x  * Populations based on bacterial numbers per cm + Based on 42 replications.  PVX 1.8 x 1 0  3  4.8 10"  5  of tissue.  9-1 x 10-  5  16  45  STEMS  TUBERS  35  30  25  20  10  0  I  40  80  120  160  200  240  280  320  +  (BACTERIAL CELLS X I0 PER CM ) 3  3  FREQUENCY  I  10  20  n m  30  (BACTERIAL  40  50  60  70  80  CELLS PER CM )  CLASSES  Fig. 3 Frequency distributions of endophytic bacterial populations i n stems and tubers of Solanum tuberosum.  3  17  significant correlation between bacterial numbers on a volume basis with fresh or dry weights i n either stems or tubers.  Nor was there a correlation  between position i n a row with bacterial number. The number of gran positive bacteria per microscope f i e l d from the stem smear i s expressed as a frequency distribution i n Figure k. bacteria per f i e l d .  Most smears contained 15 or less gram positive  But of the 180 smears examined about 3% contained 30 or  more and only 5 smears contained no bacteria.  Qualitative Assay In the i n i t i a l survey 67 isolates were obtained. generic i d e n t i f i c a t i o n i s given i n Table 2.  Their tentative  The largest number of isolates  were gram positive cocci which lost the crystal violet stain quite readily. They always appeared singly or i n irregular clusters.  They grew well on agar  containing 1:400,000 crystal violet and survived 60 C for 30 min.  These  characteristics plus the fact that they were obtained from a soil-plant environment led to the conclusion that they belong to the genus Microccocus (52).  The Pseudomonas isolates were identified on the grounds of fluorescence on King's B medium, oxidative glucose metabolism, and the presence of polar f l a g e l l a ( 7 ) .  The isolates that were gram positive or gram variable,  aerobic and produced endospores were placed i n the genus Bacillus  (6).  Flavobacterium spp. were recognized by their yellow non-water soluble pigment, lack of motility, and lack of acid production from glucose ( 7 ) . Xanthomonas isolated was similar to the Pseudomonas spp. i n a l l respects  The  50  -  >O  40  •  30  -  20  "  Z UJ  O UJ  U_  10 -  0  5  10  15  FREQUENCY  20  25  30  +  CLASSES  (NUMBER OF CELLS)  Fig. h Frequency distribution of number of gram positive rods found i n stem smears.  Numbers are based on the average  number of two microscope f i e l d s i n which bacteria were v i s i b l e .  Table 2 .  Generic identification of bacterial isolates from Solanum tuberosum  Genus  No. of isolates  Micrococcus  27  Pseudomonas  13  Bacillus .  12  Flavobacterium  2  Agrobacterium  1  Xanthomonas  1  Coryneforms  k  Other gram negative forms  7  20  except that i t lacked a fluorescent pigment but had a yellow non-water soluble pigment (7). One isolate was placed i n the genus Agrobacterium. It had peritrichous f l a g e l l a t i o n , fermentative glucose metabolism, and a colony and c e l l morphology which f i t t e d the genus description (7)-  A  number of other gram negative rods were isolated but could not be i d e n t i f i e d . In the comprehensive study of gram positive rods, coryneform bacteria and Bacillus spp. could be isolated from both stems and tubers of VF and PVX plants with relative ease. considered.  The Bacillus spp. were not further  A t o t a l of 31 coryneform isolates were obtained.  None of the  isolates obtained was pathogenic on either potato or tomato. The biochemical characteristics of several isolates were i d e n t i c a l . The characteristics of only the representative isolates are given i n Table 3.  Some of these isolates  were morphologically indistinguishable from Corynebacterium  sepedonicum (Fig. 5)-  Table 3Isolate No.  Motility  Some characteristics of coryneform bacteria isolated from Solanum tuberosum  Acid from glucose aerobic anaerobic  1  +  -  -  -  2  -  +  +  +  +  -  _  _  k 5  _  +  6  +  +  7  _  8  +  9  .  •10  -  -  11  +  _  12  _  1  Acid from lactose aerobic anaerobic  3  *  +  _  _ -  _  _ _  -  +  _  _  -  +  _  _  _  _  _  _  _  _  _  _  -  -  _ -  -  _  _  -  -  Indole Cellulose Product. Digestion  +  _  _  _  Gelatin Hydrolysis  +  +  _ -  Nitrate reduced  -  -  -  +  +  Acid Fast  +  +  -  +  +  _ _  _  -  -  _  +  -  +  * Known culture of Corynebacterium sepedonicum  ro H  22  Fig. 5  Comparison of the morphology of Corynebacterium sepedonicum  (A) with a non-pathogenic coryneform (B) isolated from Solanum tuberosum.  (Phase contrast x5000).  23  DISCUSSION The presence of endophytic bacteria i n healthy potatoes has been previously demonstrated (22, 38, 5 0 , 5 6 ) . to quantify the endophytic bacterial f l o r a .  This study i s the f i r s t attempt Since only aerobic conditions  and one type of medium was used although a l l organisms from preliminary work grew readily on i t , the true bacterial count may be somewhat higher than the results indicate i f anaerobic and more fastidious organisms were present. Nevertheless, the bacterial counts should be a r e l a t i v e l y accurate estimate of the endophytic bacterial distributions i n a given population of Solanum tuberosum. The frequency distributions show that the majority of plants have a comparatively low bacterial population, although about 25% have a higher one. Reasons for the large amount of variation i s not known. Environmental factors such as external injury and the onset of senescence probably play an important role i n predisposing the plants to bacterial colonization. The recent development of virus-free potatoes presented a unique opportunity to study plants obtained from a s t e r i l e parental clone. Virusfree potatoes have been developed from the meristematic tips of a x i l l a r y buds of heat-treated plants i n s t e r i l e nutrient solution (55) and subsequently propagated by stem cuttings ( l l ) . Because the a x i l l a r y buds remained s t e r i l e in culture i t i s assumed that the plants were i n i t i a l l y s t e r i l e . in these plants must have entered during the growing season. the root entry hypothesis of H o l l i s ( 2 2 ) .  Any bacteria  This also supports  One would, therefore, expect the  endophytic bacterial f l o r a to comprise common s o i l inhabitants. to be so i n the general survey of bacterial genera (Table 2 ) .  This proved Species of a l l  24 these genera are commonly found i n s o i l ( 2 ) . The isolates identified by other workers (22, 3 8 , 50) i s consistent with these results.  They also  obtained Bacillus and Arthrobacter (Bacterium) species. Unfortunately the coryneform bacteria could not be identified as to genus. This i s due mainly to the fact that habitat features prominently in the c l a s s i f i c a t i o n of members of this group ( 3 l ) . The isolated coryneforms were aerobes and from a plant - s o i l habitat.  Therefore, they could possibly  belong to the genera Arthrobacter, Brevibacterium, Cellulomonas, Corynebacterium, or Mycobacterium ( 7 ) . The genera Cellulomonas and Mycobacterium could be excluded because the isolates did not digest cellulose and were not acid fast (15, 2 6 ) . Some of the isolates ( 1 , 4 , and 12 i n Table 3) produced a coccoid stage i n older cultures and therefore probably belong to the genus Arthrobacter ( 1 3 ) .  The other isolates then should belong to either Brevi-  bacterium or Corynebacterium. Brevibacterium contains species of gram positive rods some of which have a coryneform morphology but f i t into no other genus (27, 5 7 ) .  The genus  Corynebacterium was f i r s t introduced to contain animal pathogenic forms such as the diphtheria bacillus (37), but plant pathogens and two saprophytic species have been included since then ( l 4 , 1 6 ) . However, taxonomists agree that at least the plant pathogens require an appropriate new taxon because of their marked difference from the original type species (±5, 5 7 , 59)-  Thus the non-  pathogenic coryneforms isolated from potato can not be placed i n any genus until-taxonomists compile sufficient information to classify the saprophytic coryneforms ( 5 7 ) .  25  The presence of endophytic coryneform bacteria i s especially significant in bacterial ring rot diagnosis by the stem smear test.  One assumption upon  which t h i s test i s based i s that the bacterial f l o r a i n the stem reflects that i n the tuber. However, t h i s study has f a i l e d to find a s t a t i s t i c a l l y significant correlation between the number of bacteria i n stems and tubers of the same plant. Another assumption i s that the presence of short gram positive rods i n stem smears i s indicative of infection by C. sepedonicum.  In Canada,  seed potatoes are considered suspect i f any short gram positive rods are found in stem smears.  This study has shown that gram positive rods, including those  with dimensions of C_. sepedonicum, can be found i n almost a l l stem smears of healthy plants, i f a careful search i s carried out (Fig. k).  When these gram  positive rods were isolated i n pure culture they were found to be non-pathogenic and biochemically different from C. sepedonicum (Table 3) although morphologically they were indistinguishable from the ring rot organism (Fig. 5 ) . Some of the isolates represented by number 12 (Table 3) were biochemically similar to C_. sepedonicum but had a coccoid stage i n older cultures and thus probably belong i n the genus Arthrobacter.  These results explain why C. sepedonicum  could not be isolated from and symptoms did not develope after several generations i n potatoes suspected of having bacterial ring rot on'.the basis of the stem smear test.  26  SUMMARY The endophytic bacterial population i n Solanum tuberosum L. stems and tubers of both virus-free and virus-infected plants was studied.  Bacteria  were found i n stems and tubers but there was a wide variation i n occurrence between plants.  No significant difference i n bacterial population was found  between virus-free and virus-infected plants, nor was there a significant correlation between populations i n stems and tubers of the same plant. Species of Micrococcus, Pseudomonas, Bacillus,\ Flavobacterium, Xanthomonas, Agrobacterium, coryneforms, and other unidentified genera" were found. Some of the coryneforms were morphologically indistinquishable from Corynebacterium sepedonicum but were found to be biochemically different.  The results of this  study questions the basic assumption made i n the stem smear test for bacterial ring rot, namely that any gram positive rods i n the smear suggests the presence of C_. sepedonicum.  27 LITERATURE CITED 1.  Adler, J . 1966.  Chemotaxis i n bacteria.  Science  2.  Alexander, M. I 9 6 I . Introduction to S o i l Microbiology. John Wiley and Sons, Inc. New York.  3.  Baribeau, B. and Marcotte, M. 1968. Methode de depistage de l a f l e t r i s sure bacterienne de l a pomme de terre, Corynebacterium sepedonicum Spieck et Koff. Phytoprotection 49:110-113.  4.  Barnes, E. H. 1965. Bacteria on leaf surfaces and i n intracellular leaf spaces. Science 147:1151-1152.  5-  Blenden, D. C. and Goldberg, H. A. 1965. Leptospira and f l a g e l l a . J . Bact.  153:708-716.  Silver impregnation stain for 80:899-900.  6.  Bradbury, J . R. 1970. Isolation and preliminary study of bacteria from plants. Rev. PI. Path. 49:213-218.  7.  Breed, R. S., Murray, E. G. D. and Smith, N. R. 1957- Bergey's manual of determinative bacteriology. 7th Edition. London, B a i l l i e r e , Tindall and Cox.  8.  B.C. Gazette, Part I I . May 14,  9.  Cameron, H. R. 1970.  P  1959-  178-179-  Pseudomonas content of cherry trees.  Phytopathology  60:1343-1346. 10.  Canada Gazette, Part I I , Vol.  103-  No. 10 May 28,  I969.  p  658-67O.  Cole, E. F. and Wright, N. S. I967. Propagation of potato by stem cuttings. Amer. Potato Jour. 44:301-304. 12. Collins, C. H. 1964. Microbiological methods. 2nd Edition. Butterworths, London. 11.  13- Conn, H. J . and Dimmick, I. 1947. S o i l bacteria similar i n morphology to Mycobacterium and Corynebacterium. J . Bact. 54:291-303. 14.  Crowle, A. J . I962. Corynebacterium rubrum nov. spec, a gram positive non acid-fast bacterium of unusually high l i p i d content. Antonie van Leeuwenhoek. J . Microbiol, and Ser. 28:183-192.  15. Da Silva, G. A. N. and Holt, J . G. 1965. Numerical taxonomy of certain coryneform bacteria. J . Bact. 90:921-92716. Dias, F. and Bhat, J . V. 1962. A new levan producing bacterium Corynebacterium laevaniformans nov. spec. Antonie van Leeuwenhoek. J. Microbiol, and Ser. 2 8 : 6 3 - 7 2 . 17. Diaz-Palanco, C., Smith, S. H. and Hanock, J . G. 1969. Effect of virus infection on stem rot squash caused by Fusarium solani f. sp. cucurbitae. Phytopathology 5 9 1 8 - 2 2 . :  28  18. Farley, J . D. and Lockwood, J . L. 1964. Increased susceptibility to root rots i n virus-infected peas. Phytopathology 54:1279-1280. 19. Frobisher, M. 1968. Fundamentals of Microbiology. 8 t h Edition. W. B. Saunders Company. 20. Glick, D. P., Ark, P. A. and Racicot, H. N. 1944. Outline of procedure for the diagnosis of bacterial ring rot of potatoes. Amer. Potato. Jour. 21:311-314. 2 1 . Graham, D. C. and Harper, P. C. 1967. Potato black-leg and tuber soft rot. Scottish Agriculture 46:68-74. 2 2 . H o l l i s , J . P. 1951- Bacteria i n healthy potato tissue.  Phytopathology  41:350-366.  23. Hopen, H. J . and De Zeeuw, D. J . I 9 6 2 . Reduction of susceptibility to cucumber scab by cucumber mosaic virus. Plant Disease Reptr. 46:93-97.  24. Hsu, S. T. and Dickey, R. S. 1972. Comparative growth of Xanthomonas phaesoli and Xanthomonas vesicatoria and development of symptoms i n bean and tomato leaves. Phytopathology 62:329-332. 2 5 . Ingram, M. and Riches, J . P. P. 1951. The preparation of s t e r i l e carrot discs for prolonged physiological experiments. New Phytol. 50:76-83.  26. Jensen, H. L. 1934. Studies on saprophytic mycobacteria and corynebacteria. Proc. Linnean Soc. N. S. Wales 5 9 1 9 - 6 l . ;  27. Jensen, H. L. 1966. Some introductory remarks on the coryneform bacteria. J . Appl. Bact. 2 9 : 1 3 - 1 6 . 28. Jones, E. D., Martinson, C. A. and Foley, E. S. 1968. Susceptibility of virus X-free potatoes to Fusarium roseum "Avenaceum" tuber rot. Amer. Potato Jour. 45:436-444. 29. Kado, C. I. and Heskett, M. G. 1970. Selective media of isolation of Agrobacterium, Corynebacterium, Erwinia, Pseudomonas, and Xanthomonas.  Phytopathology  60:969-976.  3 0 . Katznelson, H. and Sutton, M. D. 1956. Laboratory detection of Corynebacterium sepedonicum, casual agent of bacterial ring rot of potato. Can. J . Bot. 34:48-53. 3 1 . Keddie, R. M., Leask, B. G. S., and Grainger, J . M. 1966. A comparison of coryneform bacteria from s o i l and herbage: C e l l wall composition and nutrition. J . Appl. Bact. 2 9 : 1 7 - 4 3 .  29  32. K e i l , H. L. and van der Zwet, T. 1972. Recovery of Erwinia amylovora from symptomless stems and shoots of Jonathan apple and Bartlett pear trees. Phytopathology 62:39-4-2. 33- Kennedy, B. W. 1969. Detection and distribution of Pseudomonas glycinea i n soybean. Phytopathology 59:l6l8-l6±9. 34. King, E. 0 . , Ward, K. and Raney, D. E. 1954. Two simple media for the demonstration of pyocyanin and fluorscin. J . Lab. and C l i n . Med.  44:301-307.  35. Kivilaan, A. and Scheffer, R. P. 1959. Detection, prevalence, and significance of latent viruses i n Pelargonium. Phytopathology 49:282-286.  36. Leben, C. 1965. Epiphytic micro-organisms i n relation to plant disease. Annu. Rev. Phytopath. 3:209-230. 37. Lehmann, K. B. and Neumann, R. 0. 1896. Bakteriologisch Diagnostik. Munich: J . F. Lishmann. 38. Lutman, B. F. and Wheeler, H. E. 1948. Bacillus megatherium De Bary from the i n t e r i o r of healthy potato tubers. Jour. Wash. Acad. Sciences 38:336-340. 39. Matta, A. 1971. Microbial penetration and immunization of uncongenial host plants. Ann. Rev. Phytopath. 9:387-410. 40. Mulder, E. G., Adams, A. D., Antheunisse, J . , Deinima, M. H., Waldendorp, J. W. and Zevenhuizen, L. P. T. M. 1966. The relationships between Brevibacterium linens and bacteria of the genus Arthrobacter. J. Appl. Bact. 2 9 : 4 4 - 7 1 . 41. Muller, R. 0 . and Munro, J . 1951- The reaction of virus-infected potato plants to Phytophthora infestans. Ann. Appl. B i o l . 38:765-773. 42. Nagaich, B. B. and Prasad, B. 1971. Interaction between Alternaria solani and potato viruses X and Y. Indian J . Exp. B i o l . 9 8 8 - 9 0 . :  43. Nelson, G. A. 1971. Comparison of inoculation methods on ring rot development i n potatoes and virulence of cultures of Corynebacterium sepedonicum. Proc. Can. Phytopath. Soc. 3 7 : 1 9 . 44. Perombelon, M. C. M. and Lowe, R. 1971. Seventeenth Annual Report, 1970. Scottish Horticultural Research Institute 6 9 . 45. Perotti, R. 1926. On the l i m i t s of biological inquiry into the s o i l . Proc. Inter. Soc. S o i l Sci. 2 : l 4 6 - l 6 l . 46. Racicot, H. N., Savile, D. B. 0 . , Conners, I. L., 1938. Bacterial wilt and rot of potatoes - some suggestions for i t s detection, v e r i f i cation, and control. Amer. Potato Jour. 15:312-318.  30  47. Russell, G. E. 1965. The effect of Alternaria spp. on leaves of sugar beet infected with yellowing viruses. Ann. Appl. B i o l . 56:111-118.  48. Samish, Z. and Dimant, D. 1959- Bacterial population i n fresh healthy cucumbers. Food Manuf. 34:17-20. 49. Samish, Z., Etinger-Tulczynska, R. and Bick, M. 1961. Microflora within healthy tomatoes. Appl. Microbiol. 9 2 0 - 2 5 . :  5 0 . Sanford, G. B. 1948. The occurance of bacteria i n normal potato plants and legumes. S c i . Agr. 2 8 : 2 1 - 2 5 . 5 1 . Sharpe, A. N. and Kilsby, D. C. 1971. A rapid inexpensive bacterial count technique using agar droplets. J . Appl. Bact. 34:435-440. 5 2 . Skerman, V. B. D. 1967. A Guide to the Identification of the Genera of Bacteria. 2nd Edition. The Williams and Wilkins Company. 5 3 . Smith,\ M. A. and Ramsey, G. B. 1947. Bacterial l e n t i c e l infection of early potatoes. Phytopathology 37:225-242. 54. Snieszko, S. F. and Bonde, R. 1943- Studies on'.the morphology, physiology, serology, longevity, and pathogenicity of Corynebacterium sepedonicum. Phytopathology 33=1032-1044. 5 5 . Stace-Smith, R. and Mellor, F. C. 1968. Eradication of potato viruses X and S by thermotherapy and a x i l l a r y bud culture. Phytopathology 58:199-203.  56. Tervet, I. W. and H o l l i s , J . P. 1948.  Bacteria i n healthy plant tissue.  Phytopathology 3 8 : 9 6 0 - 9 6 7 .  57- Veldkamp, H. 1970. Saprophytic coryneform bacteria. Ann. Rev. Microbiol. 24:209-240.  5 8 . Walker, J . C. 1957- Plant Pathology. Company, Inc. New York.  2nd Edition.  McGraw-Hill Book  59« Yamada, K. and Komagata, K. 1970. Taxonomic studies on coryneform bacteria. I I Principle amino acids i n the c e l l wall and their taxonomic significance. J . Gen. Appl. Microbiol. 16:103-113.  31  Part B The Inhibition of Potato Pathogens by an Endophytic Bacillus Sp.  LITERATURE REVIEW INTRODUCTION The presence of bacteria i n healthy plant tissue has been well established ( l l , 1 9 , 4 4 , 4 5 , 46, 5 6 ) .  However, almost no work has been  done on the possible significance of the endophytic microflora.  Endophytic  bacteria could have diverse effects on the growth and development of the host plant.  For example, bacteria may increase the absorption of ions by  roots ( 3 ) , increase IAA production (31, 6 0 ) , affect phosphatase a c t i v i t y (42), and affect response to infection ( 3 5 ) .  Only the l a t t e r item l i e s  within the scope of t h i s review.  Biological control of plant diseases Although b i o l o g i c a l control i n plant pathology i s not a new concept i t s actual application i s almost negligible. The induction of resistance by heat-killed c e l l s (26, 3 3 ) , and similar induction by bacterial extracts has been reported (48).  This has led to the speculation that saprophytic  endophytes may also trigger such a resistance mechanism ( l 6 , 3 5 ) . At the present time b i o l o g i c a l control with antagonistic species has been investigated most extensively.  Avirulent strains of phytopathogenic  species may be antagonistic toward pathogenic strains and thus protect the host.  Kelman and Averre (23) reported the influence of avirulent c e l l s of  Pseudomonas solanacearum on the severity of bacterial w i l t .  This antagonism  32  may i n part be due to increased production of IAA by the avirulent c e l l s to which the virulent c e l l s are more sensitive ( 2 ) .  Similarly apple stem  tissue becomes resistant to infection by Erwinia amylovora after being inoculated with an avirulent strain ( l 6 ) .  Nelson and Semeniuk (38) report  the inhibition of Corynebacterium insidiosum by an antagonistic variant, in this case an inhibitor substance was p a r t i a l l y purified. Antagonistic bacteria i n the phyllosphere have also been i n v e s t i gated.  This literature prior to 1965 has been reviewed by Leben ( 2 9 ) .  Epiphytic bacteria decrease plant disease losses by inhibiting the pathogenic organism and thus preventing infection.  A short gram negative rod which  produced an antibiotic on cucumber leaves was effective i n control of cucumber anthracnose (Colletotrichum lagenarium) ( 2 8 ) .  Later i t was also found to  control early blight (Alternaria solani) of tomato and northern leaf blight (Helminthosporium turticum) of corn ( 3 0 ) .  Similarly, several epiphytic  Bacterium isolates inhibited the f i r e blight organism, Erwinia amylovora ( l 4 ) . In the l a t t e r case, increased acidity due to the antagonistic organism may have caused the protective effect. produce an antibiotic.  However, one of the Bacterium isolates did  Bacterial canker i s inhibited by a saprophytic Erwinia  sp. from the leaf surface of cherry trees ( 1 0 ) .  Some strains of Pseudomonas  fluorescens are antagonistic toward P. phaseolicola ( 5 5 ) .  Several saprophytic  bacteria from blight-infected rice leaves retarded symptom development when inoculated concurrently with Xanthomonas oryzae (^9).  Again, an  antagonistic  substance was detected but not characterized. Not only are epiphytic bacteria effective i n providing protection against bacterial diseases but also against fungal and v i r a l infections.  For  33  example, bacteria on the leaf surface i n h i b i t s germination of Botrytis cinerea spores on chrysanthemum (6) and lettuce ( 6 l ) . Other examples include the i n h i b i t i o n of Rhizoctonia solani on lettuce ( 6 l ) , necator on grape (25),  Uncinula  and Botrytis cinerea on beet leaves ( 5 ) . Evidence  of a non-pathogenic bacterium affecting a virus disease i s reported i n the Bacillus uniflagellatus - TMV interaction ( 3 ^ ) . This organism not only reduces TMV l o c a l lesions but also v i r a l content. The l i t e r a t u r e thus reveals that there are two principal ways i n which bacteria may control plant diseases. inducing a protective host response. the pathogenic organism.  The f i r s t i s by activating or  The second i s by actively i n h i b i t i n g  Antibiotics are often implicated as the inhibitory  agent.  Antibiotics i n plant pathology Species of Bacillus and Streptomyces are the chief source of antibiotics used i n the control of plant disease.  Although antibiotics theoreti-  cally could be as useful i n controlling plant infections as they are i n animal and human infections various p r a c t i c a l considerations enter the picture. Plants have a type of circulatory system (8) but i t i s far from being as effective as that i n higher animals i n dispersing the antibiotic throughout the organism.  Moreover there i s a problem of rapid inactivation of the  antibiotic once i t has been applied to the plant (53).  Despite these compli-  cations, however, there are many reports of potentially effective antiobiotics. Peach trees have been injected with oxytetracycline  to control  bacterial spot caused by Xanthomonas pruni .: (13). Streptomycin was shown to -  34  be effective i n control of halo blight (Pseudomonas phaseolicola) of beans (62), and bacterial w i l t (Erwinia chrysanthemi) of chrysanthemums ( 4 3 ) .  Stem  rust (Puccinia graminis) of wheat has been successfully controlled by cycloheximide (59) as was covered smut ( l 8 ) and powdery mildew of roses (58). Vaneomycin was found to be effective against a wide range of plant pathogenic bacteria (36).  Several Streptomyces antibiotics such as bleomycin, kasugany-  cin, bulgerin, and ablastmycin appear promising for the control of r i c e blast ( P i r i c u l a r i a oryzae) and sheath blight ( P e l l i c u l a r i a sasakii) (.17, 40, 5 0 , 5 l ) . In addition, viruses, such as tobacco mosaic virus and cauliflower mosaic virus, can be inhibited by appropiate antibiotics (20, 34, 37, 5 7 ) . Only a few of the antibiotics studied have found a commercial use. Streptomycin i s used most widely.  It i s recommended for control of blackleg  (Erwinia atroseptica) i n potatoes i n B r i t i s h Columbia (Vegetable Production Guide 1972 ( l ) .  It i s also used alone or i n mixtures with terramycin to con-  t r o l Erwinia amylovora i n hawthorn, mountain ash, pear and other members of the rose family ( 4 l ) .  Cycloheximide has been used to control white pine  .blister rust (53); powdery mildew of roses, phlox, and other ornamentals; azalea petal blight (Ovulinia azaleae); and several lawn grass diseases i n cluding Puccinia graminis f. agrostis and Curvularia lunata. used for the control of grey mold (Botrytis) of lettuce (15).  Griseofulvin i s And f i n a l l y  Bacticin i s used successfully for elimination of crown g a l l tumors caused by Agrobacterium tumefaciens and olive knot tumors caused by Pseudomonas savastanoi (47).  35  Object of project This study was undertaken to determine, the possible role of endophytic bacteria i n disease control of Solanum tuberosum L. and to elucidate the mechanism involved i n any such role.  MATERIALS AND METHODS Isolation of Bacteria and Demonstration of Antagonism Endophytic bacteria were isolated from tubers of Solanum tuberosum and identified by the methods described ( l l ) .  The 67 isolates obtained were  tested for inhibitory properties against Corynebacterium sepedonicum.  These  tests were done on a lawn of _C. sepedonicum made with a 1 ml suspension of approximately 1 x 10^ c e l l s . at four spots per plate.  The test organisms were spotted on to the plate  Media "NM" (22) was used i n these experiments and  plates were incubated at 25 C.  A. zone of no bacterial growth surrounding the  test organism indicated an antagonistic effect. One organism (Isolate 35) showing the greatest zone of inhibition was used for further study. To identify Isolate 35, dimensions and morphological characteristics were noted on gram-stained slides.  Standard bio-  chemical tests were performed by the methods previously reported ( l l ) .  In-  h i b i t i o n was also tested i n the same manner on the following potato pathogens: Pseudomonas solanacearum, Erwinia carotovora, E. atroseptica, Alternaria solani, and Phytophthora infestans.  36 Separation of the Active Substance P a r t i a l purification A technique for the p a r t i a l purification of the inhibitory substance was adapted from the method for isolating s u b t i l i n (21).  Isolate  35 was grown i n 500 ml Erlenmeyer flasks, each containing 100 ml NM, i n s t i l l culture at room temperature.  of media  After 4 days, the pH was  adjusted  to 4.5 with cone. HC1 and allowed to stand for 2 hrs at room temperature followed by centrifugation at 7,000 _g_ for 30 min.  Pellets were dried at  65 C and ground i n a mortar and pestle with 1 gm of clean fine sand.  To  the powder was added 10 ml d i s t i l l e d water and 100 ml warm 95% ethanol. The suspension was again centrifuged at 7,000 g for 30 min and the supernatant dialysed against d i s t i l l e d water for 48 hrs.  The resulting precipitate  contained the active ingredient. In later work the purification procedure was done more e f f i c i e n t l y by omitting the drying step.  Instead the f i r s t p e l l e t s were combined with an  equal amount of d i s t i l l e d water (v/v) and ground intermittantly for several minutes with a Polytron grinder.  The pH of the mixture was adjusted to 2.0  with cone. HC1 and allowed to stand at room temperature for 2 hrs.  It was  subsequently centrifuged at 7,000 _g_ for 30 min and the pellet extracted with warm ethanol i n the usual manner.  Extraction of l i p i d s The method for t o t a l l i p i d extraction was modified from Bligh and Dyer ( 7 ) .  To 100 mg of the p a r t i a l l y purified antibiotic was added 0.4  ml  37  d i s t i l l e d water, 0.5 ml chloroform and 1.0 ml methanol.  The suspension  was homogenized for 2 min i n a V i r t i s 4-5 homogenizer with the micro assembly. Another 0.5 ml chloroform was added and homogenized for another 30 sec. After being allowed to separate for a few minutes the chloroform layer containing the l i p i d s was withdrawn with a Pasteur pipette.  Detection of carbohydrates To test for the presence of carbohydrates i n the p a r t i a l l y purified antibiotic the anthrone reaction and acid hydrolysis were performed (5*0. The anthrone reaction was carried out with ko ]ig of sample to which was added 5 ml of the anthrone reagent.  The mixture was heated i n a boiling water bath  for 15 min and cooled. Absorbance was determined at 620 nm. Acid hydrolysis was carried out i n 6N HC1 for 2h hrs i n boiling water. Visible black or brownish-black humin i s formed as the result of condensation between tryptophan and any carbohydrates under these conditions (32).  Column chromatography The method of Bartley _e_t a l (4) was used for the columns of Sephadex LH-20. Sephadex LH-20 was stirred for at least h hr with aq. 90% (v/v) dimethylformamide and poured i n a 1 cm diameter glass column and packed by gravity to a height of l 6 cm. The column was then washed with aq. 90% (v/v) dimethylformamide for 6 hrs at a flow rate of 0.3 ml/min. Samples dissolved in the same solvent were added and 2 ml fractions collected. The method used for the s i l i c i c acid columns was that of Dittmer and Wells (12). A 25 mg sample of the p a r t i a l l y purified antibiotic was  38  transferred to a s i l i c i c acid column. The column had been prepared with 1 g s i l i c i c acid (100 mesh, Mallinckrodt) i n chloroform.  The column was  washed with 20 ml chloroform at a flow rate of 0.06 ml/min followed by 20 ml methanol.  The two fractions were collected separately i n bulk.  Detection of the active components Activity was detected i n the antibiotic preparations by evaporating off the organic solvents i n vacuo, suspending the dried substance i n d i s t i l l e d water, autoclaving, and placing a drop of this suspension on a fresh lawn of C_. sepedonicum. Plates were incubated at 25 C and a zone of inhibition around the antibiotic drop indicated a c t i v i t y . To test for homogeniety of a preparation thin layer chromatography ( t . l . c . ) was used.  Samples were spotted on Eastman Chromagram s i l i c a - g e l  sheets type 6o6l and separated with butanol-acetic-acid-water (4:1:1), methanolchloroform (4:1), or ethanol (99%).  Dried sheets were sprayed either with  1% (w/v) ninhydrin i n 70% ethanol or 50% (v/v) sulfuric acid and dried under an infrared heat lamp. Spots on t . l . c . were assayed for a c t i v i t y by direct assay of scrapings or their eluates. The eluates were made by three washings of the t . l . c . scrapings with the methanol-chloroform solvent. in each case by centrifugation. cribed above.  The s i l i c a gel was removed  The eluate was assayed for a c t i v i t y as des-  39  Determination of Properties of the Antibiotic Treatment with sodium dodecyl sulfate (SDS) The antibiotic was solubilized i n .001 M SDS.  The solution was  dialysed for 48 hrs to remove the SDS i n d i s t i l l e d water at room temperature.  The dialysate was subsequently assayed for a c t i v i t y .  Controls  consisted of SDS without antibiotic and antibiotic with d i s t i l l e d water instead of SDS.  Ultraviolet (U.V.) absorption spectrum The U.V. absorption spectrum was determined i n a 95% ethanol solution using a Perkin-Elmer Model 125 double beam spectrophotometer.  Infrared (I.R.) absorption spectrum One mg of the antibiotic was ground with 100 mg KBr to a fine powder with a glass rod, compressed into a pellet, and dehydrated i n vacuo. The absorption spectrum was scanned on a Beckman Infrared Spectrophotometer Model 20 A by Randy Englar (Department of S o i l Science).  Amino acid analysis A preliminary amino analysis was done on the p a r t i a l l y purified antibiotic by Dr. J . Tremaine (CDA, Vancouver). The substance was hydrolysed with 6N HC1 i n a tube sealed under vacuum and heated to 107 C for 24 hrs. Subsequent analysis was done on a Beckman Model 120 B amino acid analyzer.  RESULTS Of the 67 isolates obtained, 16 showed antagonism towards Corynebacterium sepedonicum. Twelve of these belonged to the genus Bacillus, one was a Pseudomonas sp. and the remaining three could not be identified to genus.  Isolate 35, a Bacillus sp., showed the greatest  amount of i n h i b i t i o n and was consequently chosen for further characterization of the inhibitory a c t i v i t y . Isolate 35 was a gram positive motile rod.  The vegetative c e l l s  varied i n length from 2.8 - 4 . 0 ]i and i n width from 0.7 - 0.9 usually appeared singly but occasionally formed short chains. formed readily on nutrient agar i n 3 - 4 days at 25 C. not swollen.  Cells Spores  The sporangia were  Spores (1.6 x 0.8 p,) were c y l i n d r i c a l and centrally or ter-  minally located. The isolate did not grow under anaerobic conditions and grew very poorly i n 7% NaCl.  Acid was not produced from glucose or lactose.  Tests for production of indole and reduction of nitrate were also negative. However, i t hydrolysed starch and, to a lesser extent, gelatin.  The col-  onies on nutrient agar were cream-colored, large, rough and irregular. Broth cultures formed a heavy firm p e l l i c l e with only slight turbidity. These characteristics indicate that isolate 35 was a strain of B. s u b t i l i s . In addition to inhibiting growth of C_. sepedonicum Isolate 35 also inhibited Pseudomonas solanacearum, Erwinia atroseptica, E. carotovora, Alternaria solani, and Phytophthora infestans (Fig. l ) . A metabolic by-product was implicated as the active agent when an agar disc aseptically removed from the inhibition zone was able to prevent growth of _C. sepedonicum. Several purification techniques used for Bacillus  Fig. 1. The inhibition of several potato pathogens by a Bacillus sp. isolated from healthy potato tubers.  A - Corynebacterium  sepedonicum; B - Erwinia atroseptica; C - E. carotovora; D Pseudomonas solanacearum; E - Alternaria solani.  h2 antibiotics were attempted.  The method used for purifying s u b t i l i n (21)  isolated an active fraction from the bacterial c e l l s .  One drop of a water  suspension caused a 3 - 4 cm wide inhibition on an assay plate (Fig. 2 ) . The p a r t i a l l y purified antibiotic was soluble i n ethanol, methanol, butanol, propanol, and acetone, but insoluble i n cyclohexane, benzene, and chloroform.  Activity was not lost after autoclaving at 121 C  for 15 min. The SDS treatment also did not destroy a c t i v i t y i n four of five replications.  The amino acids detected on one assay i n the p a r t i a l l y  purified antibiotic i s given i n Table 1. Both teste for the detection of carbohydrates were negative.  The U.V. and I.E. absorption spectra are  given i n Figures 3 and 4 respectively. T.l.c. plates of the p a r t i a l l y purified antibiotic showed three spots with a l l solvents. The Rf values were as follows: ethanol 0.23, 0.32, and 0.77; methanol-chloroform water 0.74, 0.79, and O.83.  0.49, 0.73, and 0.84; butanol-acetic acidThe f i r s t two spots i n a l l cases gave a positive  ninhydrin reaction. The third was light yellow i n color before developing and could be marked more easily after the H^SO^ spray.  Because the methanol-  chloroform solvent gave good separation most rapidly i t was used i n a l l subsequent tests. Assay of eluates from the three spots on the t . l . c . plates showed that the spots at Rf 0.73 and 0.84 with the methanol-chloroform  solvent had  biological a c t i v i t y (Fig. 5 ) . The l a t t e r spot showed the greatest amount of inhibition. eluates.  Activity was not enhanced by combining any of the three  Fig. 2.  The inhibition of Corynebacterium sepedonicum by the p a r t i a l l y  purified antibiotic from Bacillus sp. The plate shows three zones of inhibition each due to one drop of an aqueous suspension of the substance.  The lower right hand area of the plate i s the control area  having been inoculated with a drop of s t e r i l e d i s t i l l e d water.  Table 1. Preliminary l i s t of amino acids present i n the p a r t i a l l y purified antibiotic from an endophytic Bacillus sp.  Amino Acid  Ratio  Alanine  2  Aspartic acid  5  Cysteine  1  Glutamic acid  11  Glycine  1  Iso-leucine  2  Leucine  11  Lysine  2  Phenyalanine  1  Proline  3  Serine  1  Threonine  3  Tyrosine  3  Valine  k  h5  Fig. 3.  The U. V. absorption spectrum of the p a r t i a l l y  purified antibiotic measured i n 95% ethanol.  he  Fig. h.  The I. R. absorption spectrum of the p a r t i a l l y  purified antibiotic i n a KBr pellet.  Fig. 5.  Inhibition of growth of Corynebacterium sepedonicum by  two active fractions from the p a r t i a l l y purified antibiotic as separated on t . l . c . by methanol - chloroform.  The upper zones  of inhibition coincide with the eluate from spots at Rf 0.73 and lower zones from spots at Rf 0.84.  48  The other methods for separation of the three components were not successful.  The active substance was present i n the chloroform fraction of  the l i p i d extraction procedure and i n the methanol fraction of the s i l i c i c acid column. However, these fractions did not d i f f e r from the o r i g i n a l purified preparations on t . l . c . the Sephadex LH-20 column.  Moreover, no separation was achieved with  49  DISCUSSION Bacillus spp. have been an important source of antibiotics. The organism used i n this study also belongs to t h i s genus.  Isolate 35  i s probably a strain of Bacillus s u b t i l i s although the great number of variations i n the cultural characteristics exhibited by the various strains of this species makes positive identification d i f f i c u l t ( 9 ) .  This strain  differed from the species description (9) of B. s u b t i l i s i n that no acid was produced from glucose, n i t r i t e s were not produced from nitrates, and there was only poor growth i n 7% NaCl.  The negative test for acid from  glucose, however, may be due to liberation of ammonia from peptone resulting i n complete neutralization of the acid ( 5 2 ) . The inhibitory properties of t h i s organism aire due to two antib i o t i c substances. The substance of Rf value 0.73 with the methanol-chloroform solvent, was ninhydrin positive and therefore indicated a peptide.  The  evidence from the U.V. and I.E. spectra and amino acid analysis also indicates a peptide.  However, these results must be interpreted with caution due to the  presence of another ninhydrin positive spot on t . l . c . plates of the p a r t i a l l y purified substance. The amino acid analysis (Table l ) only indicates which of the amino acids may be present i n the antibiotic.  The large amount of  leucine could account for the i n s o l u b i l i t y i n water.  The 280/260 absorption  ratio i n the U.V. spectrum was 0 . 9 3 . I t indicates the presence of the aromatic amino acids, tryptophan and tyrosine. 1800 and 1100 cm amino acids.  The peaks between wave numbers  i n the I.E. spectrum are caused by the amide groups of the  I.E. spectra are often used to identify antibiotics.  Although  the active fractions were not obtained i n a pure form i t i s interesting to  50  note that the spectrum was similar to b l a s t i c i d i n A (2k), and p l u r a l l i n (39). Both are wide spectrum peptide antibiotics obtained from species of Streptomyces. The other active fraction i s a l i p i d .  The absorbance near 200 nm  in theU.V.. spectrum i s probably due to this fraction.  The l i p i d also ex-  plains why the SDS did not inactivate the p a r t i a l l y purified antibiotic. The l i p i d was not identified but the results of the s i l i c i c acid column indicates a complex l i p i d because chloroform removes the simple and methanol the complex l i p i d s (12). The l i p i d extraction procedure (7) was unsuccessful because the p a r t i a l l y purified antibiotic was insoluble i n this solvent system.  Sephadex  LH-20 has been used to separate ethanol-soluble peptides (k); however, no separation of the p a r t i a l l y purified antibiotic was achieved with i t . A l though the structure of the active components could not be determined due to d i f f i c u l t i e s i n separating the components, i t can be concluded that the inhibition i s due to both a peptide and a l i p i d . This i s the f i r s t report of the production of an antibiotic by an endophytic micro-organism.  Although _in vivo a c t i v i t y has not yet been  demonstrated, the organism has potential for disease control.  As already  noted, transport and rapid inactiviation are unresolved d i f f i c u l t i e s i n the use of antibiotics (53)• However, an antibiotic-producing, endophytic population beneficial to the host solves both problems.  The bacteria can move  through the plant and can be a constant source of antibiotic.  Such a system  may not prevent i n i t i a l infection but should prevent further progression of the pathogenic organism.  The vegetative propagation of potatoes lends i t s e l f  51  very well to such a system.  Once the desirable endophytic population has  been established i n a clone i t could be maintained indefinitely through the tuber. Such a symbiotic relationship has admittedly not been proven to be effective.  However, we are only just beginning to explore the possi-  b i l i t i e s of biological control i n plant pathology and this i s definitely one which should not be ignored.  SUMMARY Sixteen of 67 bacterial cultures isolated from healthy Solanum tuberosum plants were inhibitory toward Corynebacterium sepedonicum.  A  Bacillus sp. probably a strain of Bacillus s u b t i l i s was used for further characterization of the inhibitory a c t i v i t y .  This species was also i n -  hibitory toward the following potato pathogens: Pseudomonas solanacearum, Erwinia atroseptica, E. amylovora, Alternaria solani, and Phytophthora infestans.  An antibiotic was p a r t i a l l y purified.  Thin layer chromatography  followed by bioassay showed that two active substances were present. fraction was identified as a peptide the other as a l i p i d .  One  52  LITERATURE CITED 1.  Anon. 1972. Vegetable Production Guide. Departments of Agriculture.  2.  Abo-El-Dahab, M. K. and El-Goorani, M. A. 1969. Antagonisms among strains of Pseudomonas solanacearum. Phytopathology 59 1005-1007-  Canada and Provincial  :  3.  Barber, D. A. and Frankenburg, U. C. 1971The contributions of microorganisms to the apparent absorption of ions by roots grown under nons t e r i l e conditions. The New Phytologist 70:1027.  4.  Bartley, I. M., Hodgson, B., Walker, J . S. and Holme, G. 1972. 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