RHIZOSPHERE STUDIES IN RELATION TO VARIETAL RESISTANCE OR SUSCEPTIBILITY OF TOMATO

TO VERTICILLIUM WILT'

Abstract The density of ful~gi in the rliizosphere and the nature of fungi on the rhizo-

plane of five varieties of tomato, sonic susceptible and some resistant t o infection by Verticillium, were determined. The numbers of fungi were higher in the rhizospheres of the susceptible varieties, Bonny Best and John Baer, than in the resistant ones, Loran Blood and Moscow. However, in the rhizosphere of the resistant variety Geneva 11, the numbers of fungi did not differ appreciably from those for the susceptible varieties. Among the fungi isolated from the rhizoplane, a species of Fzlsariilwz was dominant on the varieties Bonny Best, John Baer, and Geneva 11 and Trichoderrna viride Pers. was dominant on the varieties Loran Blood and Moscow. In vitro, the rhizoplane fungi did not show antagonism, either against Verticilliz~m or amongst themselves. Nevertheless, disease intensity was reduced in vivo when roots of susceptible varieties were inoculated with Ver t ic i l l i z~?~~ in association with Trichoderma. Verticilliz~nt grew equally well in root exudates of all the varieties. Similarly, Fz~sariunt and Trichodernza grew well in the exudate of the susceptible Bonny Best and the resistant Geneva 11. Chromatographic analyses of root exudates of the different varieties, collected aseptically, revealed the presence or absence of several amino acids, glucose, and fructose.

Introduction The roots of higher plants are known to exude various substances such as

amino acids, sugars, and organic acids (2, 4, 11, 12, 16, 19). These exudations, together with the sloughed-off debris of the root cortex, provide a localized increase in metabolites in the vicinity of roots. This increase is reflected in an intense biological activity in the soil immediately surrounding the roots, as shown by the marlied increase in the numbers of fungi and bacteria in the rhizosphere. Recent reviews on the subject are provided by Lochhead (13) and Sadasivan and Subramaniail (20). Recently, in the study of root diseases, emphasis has been placed on the differential effects on the pathogen of root exudates of resistant and susceptible varieties of the host. Andal et al. (2) recorded higher numbers of amiilo acids in the exudates of a variety of rice (Oryza sativa L.) susceptible to foot rot caused by Gibberella fujikuroi (Saw.) Wr., than in one resistant to the pathogen. Buxton (5, 6) found that aqueous extracts of rhizosphere soil and root exudates of three pea cultivars differential- ly influenced the germination of spores of three physiologic races of Fusariz~m oxysporzlm f. pisi. He correlated the effects on germination with resistance or susceptibility of a host to infection by a race. In the present investigation, five varieties of tomato, three resistant and two susceptible to wilt caused b y Verticillium albo-atrzlm Reinlie and Berthold, have been studied with respect to quantitative changes in the fungal flora of their rhizosphere, the nature

'Manuscript received June 27, 1961. Contribution froin the Department of Botany, University of Toronto, Toronto, Canada.

2National Research Council of Canada Postdoctorate Fellow. Presently a t the University Botany Laboratory, Madras-5, India.

Can. J. Botany. Vol. 39 (1961)

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1748 CAN:\DI.kN JOURNAL OF BOTANY. VOL. 39. 1961

and frequency of fungi occurring on their rhizoplane, the qualitative nature of their root exudates, and the possible interaction of all these factors with the pathogen in question.

Materials and Methods The tomato varieties used in the present investigation were Bonny Best,

John Baer, Geneva 11, Loran Blood, and i\/Ioscow, the first two of which are susceptible to Vert,icillizlm wilt, while the latter three are resistant. Seed of Loran Blood and Mosco\v was obtained from Utah Agricultural Experiment Station, U.S.D.A., Logan, Utah, and seed of the remaining varieties was obtained from commercial dealers. The culture of Verticillizlnz was isolated by Dr. P. I<. Basu in this department from wilting toinato plants (3). Plants were raised in the greenhouse in wooden flats filled with garden soil composed of peat and loam (1 : 1). The soil was watered with tap water every 24 hours, approximately the same quantity being added to each flat. Flats containing unplailted soil sin~ilarly watered supplied non-rhizosphere soil. Samples of rhizosphere soil were obtained as follows: the plants were carefully removed and shaken individually to remove clumps of soil adhering to the root system. The root systems were then scissored off froin the top of plants a i d trans- ferred individually to aliquots of 100-ml sterile tap water in 250-in1 Erlen- meyer flasks fitted with rubber stoppers. Sufficient roots were collected to provide a t least a gram of soil. The flasks containing the roots were then shaken on a Burell shaker a t constant speed for 5 minutes, following which the roots were removed and the soil-water subjected to serial dilution. In this process of serial dilution, the subsequent shaliings of the soil-water were of the order of a minute each. One milliliter aliquots of the 1 : 10,000 dilution of each sample were poured into sterile plates and approximately 25 ml peptone-dextrose (15, 17) agar was added. The non-rhizosphere soil was obtained by removing and rejecting the first half-inch layer of top soil from the uilseeded flat and, a t that level, samples of approxin~ately a grain of soil were taken a t random. These were treated in a manner similar to the rhizo- sphere soil san~ples. Counts of fungi were taken on the 6th day after plating, from six replicate plates.

Studies on the rhizoplane were confined to the tap root next to the hypo- cotyl, the length of which varied from 4 in. to 2 in., depending on the age and vigor of the plant. The serial washing of roots was similar in principle to that of Harley and Miaid (S), but, instead of using screw-capped bottles, the apparatus shown in Fig. 1 was designed to permit the continuous washing of the roots. This apparatus consists of a glass container f with a capacity of approximately SO ml. I t may be disjointed a t e and has a chromel wire inesh g to hold the root segments. The outlet a t the top connects with two sterile water reservoirs b. The container f was attached to a Burell shalier running a t constant speed and equipped with an automatic timer. During each washing 40 ml of sterile water was admitted into the container, the shalier operated for the desired time, and the wash water draiiled by opening the outlet a t the bottom. The lateral outlet a', plugged with sterile cotton, served to break the vacuum a t each operation. Approximately 8-10 washings were necessary to get rid of all the extraneous soil and spore masses on the roots. The numbers

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SUBBA-RAO AND BAILEY: RI-IIZOSPHERE S T U D I E S

FIG. 1. Slictches of apparatus used. A. For continuous washing of root segments. B to E. Method of growing multiple and single plants, in two stages of operation. a , Cotton plug; a', lateral outlet; b, sterile water reservoir; c, rubber connections; d , pinchcock; e, ground-glass joint; f, container; g, chrome1 wire mesh to hold root segments; h, cup to hold the chrome1 wire mesh; i, vessel to contain the ~iutrient solutio11; j , plant(s); kl ,

inlet for nutrient; k?, inlet for aeration; kn, exit for nutrient; I , lid with an upper limb to hold the shoot; nz, orifice; n, lid; o, glass beads.

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CANADIAN JOURNAL O F BOTANY. VOL. 39, 1961

NUMBER OF WASHINGS

FIG. 2.INumbers of fungi isolated in relation to successive washings of root segments.

of fungi decreased gradually during subsequent washings and from the graph (Fig. 2) it can be seen that a t least 20-25 washings were necessary to bring the counts to a minimurn. On this basis, 50 root segments were shalien 25 times in 40 ml of water, for 5 minutes in the first wash and for 1 nzinute during each subsequent washing. The washed root seglllellts were cut into approximately 1 cm pieces and plated on peptonedextrose nutrient agar (15, 17). More than 100 fungi growing fro111 these washed roots were trans- ferred to agar slants for identification.

Root exudates were collected from plants grown in the two types of appar- atus shown in Fig. 1. These were housed in a controlled-enviro~l~nent chamber (75" F temp. and 1150 ft-c. light for 16 hours a day). Of the two types of apparatus, one was designed to culture a nu~nber of plants and the other to culture single plants, aseptically. The first type (B and C in Fig. 1) consists of a main glass vessel (i) capable of contai~ling 150 ml of nutrient solution. I t has three inlets: k l to replenish the nutrient, kz for aeration, and ks which served to withdraw the nutrient. Resting on the neck of the main vessel i is a chrome1 wire mesh g held in position by a cup h. A lid n, held loosely by cotton gauze a , closes the nlouth of the main vessel. Nutrient was added by removing the lid n, and tomato seeds which had been surface-sterilized in

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SUDBA-RAO AND B.4ILE.Y: RHIZOSPHERE STUDIES 1751

lYo sodium hypochloride for 10 minutes were placed on the chrome1 wire mesh. When the plants were a few days old, the lid was removed and a suffi- cient quantity of glass beads o was poured in to fill the spaces around the plants. The glass beads served to prevent aerial contamination and facilitated the free groxvth of the shoot system. Plants were maintained in this way for a maximum of 8 weeks. In the second set-up (D and E in Fig. I ) , for the vessel i of the first type of apparatus, a 250-ml Erlenmeyer flask was substituted and fitted with a special lid n having a basal orifice m of 2-3 mm diameter and an upper limb to hold the plant erect. A seedling, previously germinated on water agar, was gently pushed up the orifice. The lid n was removed and two glass beads o were dropped in to keep the seedling in position. Nutrient solutioll was poured into the flask and the lids n and 1 were placed in position. After a few days, the lid n was removed and more glass beads were poured around the stem to re vent aerial contamination. Plants were maintained in this way for a maximum of 9 weelis. The root system was aerated for 2-3 hours each day with filtered compressed air through the inlet k2. Direct light was prevented from falling on the roots by covering the lower part of the apparatus with black paper. The exudate was checked for bacterial contarnina- tion by plating out samples and only those free of contaminants were used for experimental study.

A minimal-strength nutrient medium of the following composition was used for growing plants, so as to avoid excessive interference of salts during paper chromatography: KC1-0.5 g, KNOB-1.0 g, KH,POa-0.5 g, R4gS04- .7H00-0.25 g, CaS0.i--0.25 g, Ca(P04)!-0.25 g, FeC13-0.02 g, and distilled water-1000 ml. The exudates were concentrated to dryness by vacuum condensation a t 30' C and taken up in 2 ml of 50% alcohol. Increasing con- centrations were spotted to detect the maximum nulnber of compounds. Amino acids were deterrnilled by two-way ascending chromatography on Wl~atman No. 1 filter paper using SOYo phenol for the first run and a mixture of n-butanol, acetic acid, and water (9: 1 : 2.5) for the second run. The devel- oped cl~romatograms were sprayed with 0.4Y0 ninhydrin in acetone. One- way ascending chromatograms were run in n-butanol, acetic acid, and water (4:1:5) and in SOYo phenol and sprayed with aniline phthalate mixture, to determine sugars.

Results Rhizosphcre

The differences in the fungal population in the rhizosphere soil of the five varieties a t three different ages are shown in Fig. 3. In all cases, the numbers of fungi were higher in the rhizosphere soil than in the non-rhizosphere soil and, in the latter, incubation had no appreciable influence on the numbers. On the other hand, with increasing age, a decrease in numbers of fungi was noticed in the rhizosphere of all the varieties. Moscow and Loran Blood had only half or less than half as many fungi as Bonny Best and John Baer. I n 14-day-old plants no marked difference between the resistant Geneva 11 and the susceptible varieties was seen. Nevertheless, the nulnber of fungi was considerably lower in Geneva 11 than in the susceptible ones during subse- quent periods.

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CANADIAN JOURNAL OF BOTANY. VOL. 39, 1961

-BONNY BEST - JOHN BAER - GENEVA I I - LORAN BLOOD - MOSCOW - NON - RHIZOSPHERE

14 2 8 5 6

AGE - DAYS

FIG. 3. Relation of age to numbers of fungi in the rhizospheres of resistant and susceptible varieties.

Rhizoplane The relative incidence of different fungi occurring on the rhizoplane of the

five varieties, a t three different ages, is shown in Fig. 4. From 70 to goy0 of the fungi on John Baer, Bonny Best, and Geneva 11 was a species of F ~ ~ s a r i u m which either was entirely absent or occurred in the 10-30yo range on the roots of Loran Blood and Moscow. On the other hand, Trichoderma viride was dominant on Moscow and Loran Blood, while only 8-10yo of fungi from John Baer was Trichoderma, which was not isolated a t all from either Boll~ly Best or Geneva 11. Other species of fungi occurred on all the varieties, in varying degrees, and were grouped under 34ucorales, Penici l l ium spp., Asper- gillz~s spp., and other fungi.

Interaction The fungi isolated fro111 the rhizoplane were grown opposite V e r t i c i l l i ~ ~ m

on Petri plates colltaining Czapeck-Dox agar and Martin's agar to see i f any antibiosis occurred. The results were negative. Similar studies revealed that the isolates from the rhizo~lane were not inutuallv antibiotic.

Studies on the gro\vth of Vertici l l iz~m in liquid cultures containing root exudates indicated no appreciable differences in the effect of exudates of the different varieties on the growth of the pathogen. With a view to studying the effect of root exudates on the growth of dominant fungi occurring on the rhizopla~ze, F ~ ~ s a r i z ~ m and Trichoderma were grown on different concentrations of root exudates of Bonny Best and Moscow. Exudate from approxinlately 120, 6-week-old plants was concentrated to 200 1111 in vacuo a t 30' C , sterilized by filtering in a Morton pyrex glass filter equipped with an ultra-fine fritted dislr, and dispensed into sterilized 250-m1 Erlenmeyer flasks containing appropriate aliquots of distilled water so as to provide 100, 50, 25, and 10yo concentrations in 25-1111 volumes. Duplicate flaslts containing the different concentrations of the exudate of both the varieties were inoculated separately with mycelial/conidial suspensions of Fz~sar i z~m and Tricltoderma. The flasks

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SUBBA-RAO AND BAILEY: RHIZOSPHERE STUDIES

1 BONNY BEST k JOHN BAER

14 2 8 5 6 AGE IN DAYS

2 8 AGE IN DAYS

14 2 8 5 6 AGE IN DAYS

14 2 8 5 6 AGE IN DAYS

95 F LORAN BLOOD

FUSARIUM SP. TRICHODERMA VlRlDE

- PENlClLLlUM SPP. - ASPERGILLUS SPP. - MUCORALES - OTHER FUNGI

14 2 8 5 6 AGE IN DAYS

FIG. 4. Fungal flora of the rhizoplane of resistant and susceptible varieties a t different age.

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1754 CAWADI.-\N JOURNAL OF UOT.4NY. VOL. 39, 1961

were kept uindcr observation for 3 xveelts. The two fungi grew equally well in the exudates of both the varieties of tomato and showed a gradient in growth with increasing concentrations of the exudates (Figs. 5 arid 6).

A m i n o Ac ids and Sugars in Exz~dates During tlne earlier part of thc studies, exudates from 2- to 8-weel;-old

plants of the two varieties Bonny Best and Geneva 11 were talten up for investigation. Esudate from approxi~nately 120 plants grown in the multiple- plant apparatus was condcnscd and analyzed. The varieties John Baer, Loran Blood, and Moscow were included in the study a t the 8-week age level. T o obtain the exudate a t this age, all live varieties were grown in the single-plant apparatus; exudate from a set of six plants of each variety con- stituted a sample for analysis. Considering the relation of age to amino acid content (Table I ) , it will be seen that the number of amino acids was liiglner in Geneva 11 a t 2, 3, and 6 wcclis than in Bonny Best of the same age; during the 4th and 5th weelis the nulnber and nature of amino acids in both varieties were similar. Glucose and fructose were not detected during the 2nd ailcl 3rd weelts but were detected in subsequent weelts. By comparing the exuclate pattern of all the varieties a t the 8th weel;, it will be seen that John Baer had the maximum number of amino acids and Loran 131ood a~zd R/Ioscow had the minin~um, while Bonny Best and Geneva 11 were internzediate, having five and six amino acids respectively. Glucose was recorded in all the varieties while fructose was detected in all except Loran Blood and i\/Ioscow.

Infection Studies While the rhizoplane fungi failed to exhibit a~ltagonism in vitro, the possibil-

ity of the fungi operating i n vivo with Vert ici l l iz~m remained open. Hence, pathogenicity of TTerticidlium to susceptible tomato was tested with and with- out the presence of P u s c ~ r b m or Trichoderma. Root-dipping techinque (3) was followed. Cultures of Ver t ic i l l i z~m, F t l sn-r i~~m, and Trichoderma were grown 011 100 ml Czapeck-Dox liquid medium in 500-1111 flaslcs for 6 weeks. 'The mycelia were waslled in water by rcpcatecl centrifugation, macerat-ecl in a Waring blenclor, and suspencled in aliquots of distilled water. Six-week-old susceptible Bonny Best ancl John Baer plants, raised in sterilized soil, were waslled lvell in tap water ancl dipped i l l fungal susperlsion of either F ~ ~ s n r i l ~ r n or Trichoderma for 8 hours ancl then transferred to fungal suspension of TTerticillizlm for 4 IIOLI~S. Plants exposed to each of the three fungi separately servecl as controls. The treated plants wcrc transplanted to pots containing Perlite and fed with Crone's nutrient. They were incubated in the greenhouse a t 70-80" F. Vein-clearing of the leaves was the earliest symptom of tlne disease (Fig. 5). At the end of 10 weelts the plants were classified according to symptom expression as follows: 1 , healthy; 2, leaves showing vein-clearing; 3, leaves showing chlorosis; 4 , leaves flaccid and drooping; and 5, plants dead. The results are shown in Table 11. The number of cases showi~ig vein- clearing, flaccidity, and drooping of leaves was reduced in plants infected with TTerticillium in association with Triclzodermz to such an extent that the total number of plants showing one type of symptom or the other uras only half that of the ones infected with Vert ici l l iz~m alone or in coinbinatio~n with

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FIG. 5. A and R. The two apparatuses for aseptic collection of root exudates. C and D. Typical vein-clearing synlptoms on susceptible plants infected by Vcrticilliz~~tz; C shows the symptom in its incipient stage in a young plant, while 1> shows varying degrees of vein-clearing in an adult plant.

Subba-Rao and Bailey-Can. J. Botany

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FIG. 6. A. Effect of root e x ~ ~ d a t e of the variety Bonny Best or1 the growth of F ~ ~ s a ~ i ~ ~ t i t sp. B. The effect of root esudate of thc variety Moscolv on the gro\vtl~ of Tr i c l~oder i va viride. In A and B, the flasks fro111 left t o right contain 100, 50, 25, and 10% co~lcentration of exudates.

Subba-Rao and Bailey-Can. J. Botany Can

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SLiBBtl-R:\O A N D B-XILEY: RI-IIZOSPIIERE STUDIES

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1756 CANADIAN JOURNAL OF BOTANY. VOL. 39, 1961

TABLE I1 Summary of results of inoculation experiments*

Readings - --

Symptom group A(40) B(45) C(44) D(46) E(45) F(44)

1. Normal 4 5 5 6 2 9 2 7 2. Leaves showing

vein-clearing 6 4 8 10 1 2

3. Leaves showing chlorosis 17 21 17 16 11 13

4. Leaves flaccid and drooping 10 12 13 12 4 2

5. Plants dead 3 3 1 2 0 0

*A = Bonny Best + Verlicillirim, B = Jolln Baer + Vcrlicilliz~tn. C = Bonny Best + F~~sar i r i r~z + Verlicil- lirctn, D = John Baer + Ftcsarilrtn + Verlicillirim, E = Bonny Best + Trichoderara + Verlicillizinz, F = John Baer + Trichoderma + Vcrlicilliunr. Numbers in parentlleses indicate the total number of plants.

Fusarium. Plants infested with either Fzisarium or Trichoderma alone did not show ally symptoms.

In an experiment designed to test the pathogenicity of the isolate of Verti- cillium used in the present study, 8-week-old Boilny Best and John Baer plants grown aseptically in the single-plant set-up (D and E in Fig. 1) were inoculated with Verticillium by the root-dipping technique, transplanted illto sterilized soil, and incubated in the greenhouse a t 70-80° F. Eight weeks after transplanting, vein-clearing of leaves appeared as the first syinptoill of disease (Fig. 5) in all the plants and was expressed in an acropetal order. The fuilgus was illvariably recovered from roots and stems of these plants.

Discussion Although the inlportance of the rhizosphere has beell recognized from the

time of Hiltiler (lo), its potentialities in the field of root disease, especially in relation to the nletabolic products exuded by the root system, has attracted the attention of soil inicrobiologists and plant pathologists inore recently (1, 5, 6, 14, 18, 23). The gross differences in the populatioll of fungi between suscentible and the resistant varieties observed in the wesent studv collfirnzs the earlier finding that varieties susceptible to many root diseases generally harbor higher numbers of mici-oorganisnzs in their rhizosphere than do resistant ones (1, 14, 18, 23). Predoininance of a species of Fzisariz~m among the fungi occurriilg on the rhizoplane of all the susceptible varieties ancl Trichoderma on two of the three resistant varieties suggested an inquiry into the part played by these fungi ill disease incidence. Notwithstanding the failure of the rhizoplane fungi and Verticillium to exhibit antagoilism in vitro, the significant protection offered by Trichodermu in reducing infection rate in vivo on susceptible plailts is suggestive ill view of the fact that this genus has been regarded by numerous investigators fro111 the time of 'CVeindling (24) as a factor governiilg the activity of pathogenic fungi ill the soil (26).

Symptoms such as epinasty, vein-clearing, and fluoresceilce of plant parts have served for the detection of infection in its incipient stage in inally hadro-

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SUBBA-R.40 AND BAILEY: RI-IIZOSPHERE STUDIES 1757

ixycoses of plants other than Verticilliz~m wilt (7, 21, 22, 25). During our iilfectioll experiments on 2- and 4-month-old plants inoculated by the root- clipping technique, we observed vein-clearing syinptoms of leaves as the first sign ol the disease; in the 4-month-old plants, this symptom progressed acropetally and was followed by chlorosis of i~itervei~lal areas leading to wilt.

Amino acids appeared to constitute the hulk of the substa~lccs exuded by roots. Judgiilg from the ainino acid pattern of exudates of different varieties of 8-week-old plants, it might be inferred that the resistailt varieties Loran Blood and ~\/IOSCOW exuded fewer ainiilo acids than did the susceptible vari- eties. Elowever, comparative studies of resistant Geneva 11 and susceptible Uonliy Best a t weel;ly intervals showed the occurrence of higher numbers of amino acids in the former than in the latter, especially during the earlier weeks. These facts suggest that, while amino acicls inay conceivably play a major role in the nutrition of the rhizosphel-e organisms, a more detailed study regarding the specific role ol each of these amino acids would be neces- sary before one could ascribe ally significailce to them with respect to resist- ance or s~~sceptibility. The absence of sugars in the exudates during the first three weelts and the presence of oilly glucose aild fructose during subsequent weeks suggest that these easily assilnilable carbohydrates come into the picture of rhizosphere only after the plants are fairly advanced in age.

References 1. AGNI~IOTI. IR~DU, 17. 1957. The density of the rhizosphere rnicroflora of pigeon-pea (Ca-

jalzzis cginn L. Millsp.) in relation to wilt ca~ised by filsarizlln zidzlm Butler. Natur- \\,issenschaften. 18, 497.

2. AYDAI*, R., B I ~ I U ~ ~ A ~ E S ~ ~ A R I , Ii., and SUUB:\-RAO, N. S. 1956. Root exudates of paddy. Nature, 178, 1063.

3. B ~ s u , P. I<. 1961. l~ertzcilliz~nz disease of strawberries. Can. J. Botany, 39, 165-196. 4. BIIG\~:\XESW:\RI, I<. aiiil Suuna-R;\o, N. S. 1957. Root exudatcs in rclation to the rhizo-

spliei-e elTcct. Proc. Indian Acad. Sci. 45, 299-301. 5. Bux.~.ox, 1.3. IV. 1957. Sollie elrccts of pea root exuclatcs on physiologic races of F z ~ s n r i ~ ~ n z

oxysporzlm f . pisi (Linf.) Snyder ruid I-Iansen. Trans. Brit. hiIycol. Soc. 40, 145-154. 6. Bnslror, E. W. 1957. DilTercntial rhizosphere clfccts of three pcn cultivars on physiologic

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