ROOT EXUDATION IN COWPEA AND SORGHUM AND THE EFFECT ON SPORE GERMINATION AND GROWTH OF SOME SOIL FUSARIA

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1 New Phytol. (1978) 80, ROOT EXUDATION IN COWPEA AND SORGHUM AND THE EFFECT ON SPORE GERMINATION AND GROWTH OF SOME SOIL FUSARIA By V. S. AYO ODUNFA Department of Botany, University oflbadan, Ibadan, Nigeria {Received 31 October 1977) SUMMARY Exudation patterns in cowpea and sorghum seed and seedlings were studied using a filter paper technique. Profuse exudation was observed from cowpea seeds but this decreased considerably 2 days after germination. Root tips of primary, lateral, and adventitious roots were found to be the major sites of exudation but the older parts of seedling roots also exuded significant quantities of amino acids. Exudates from the roots of both species enhanced the germination of conidia of four Fusarium species previously isolated from the rhizosphere and rhizoplane. The germ tubes and some conidia were lysed by cowpea exudate in 48 h. Mycelial growth in liquid culture was enhanced in cowpea root exudate medium but was scanty in sorghum exudate; the presence of antifungal substance was suspected in the latter. The significance of the results obtained in relation to microbial root colonization and pathogenesis is discussed. INTRODUCTION Root exudates are one of the important major factors in the colonization of roots by soil microorganisms. They contribute significantly both to the attraction and establishment of the rhizosphere fungi, root pathogens and soil bacteria. Unfortunately, knowledge is still inadequate both on the sites of exudation and the exact role of the nutrients in the germination and growth of soil microorganisms. Considerable differences have been observed in the effects of root exudates on spore germination and mycelial growth; these effects vary from general stimulation, specific growth stimulation, to inhibition (Schroth and Hildebrand, 1964). The present study is designed to identify the major sites of root exudation and their effect on fungal spore germination and mycelial growth. A filter paper method employed in this study was found suitable for observing sites of exudation at the early stages of germination. The experimental plants were cowpea (Vigna unguiculata (L.) Walp.), and sorghum {Sorghum bicolor (L.) Moench.). The fusaria are an appropriate choice as test species since they are amongst the most ubiquitous fungi in the soil. Their spores are usually dormant in the soil but respond quickly to organic substrates. Many are saprophytes or facultative parasites and occasionally fastidious pathogens of plants. The four Fusarium species found predominant in the rhizospheres and rhizoplanes of cowpea and sorghum (Odunfa, 1975) are: (1) Fusarium solani (Mart.) Sacc. var 1; (2) F. solani (Mart.) Sacc. var 2; (3) F. oxysporum Schlecht.;(4)F. semitectum Berk, and Rav. MATERIALS AND METHODS Seeds of Cowpea, Black Eye cv. Mala and Sorghum of the Guinea race were used X/78/ $ Blackwell Scientific Publications 607

2 608 V. S. AYO ODUNFA Detection of sites of exudation. Apparently healthy seeds with intact seed coats were selected. They were surface-sterilized by immersing in 20% Milton sterilizing fluid containing (w/w) 1% sodium hypochlorite and 16.5% NaCl. Ten seeds were removed for immediate investigation while the others were plated on sterilized moistened filter papers in Petri dishes on the laboratory bench at room temperature. Each day, up to 6 days after plating, a sample of ten seeds was taken and tested for the exudation of substances. Germination occurred 2 days after plating in both species. Selected seedlings were picked with a sterilized pair of forceps to avoid contamination and placed 5 cm apart near the edge of filter papers inside sterile Petri dishes. Whatman No. 3 filter paper proved to be the most suitable as it is highly absorbent. Sterilized, bleached, non-absorbent cotton wool was laid on top to press the roots down and encourage them to grow flush with the filter paper. The paper was moistened with sterile distilled water. The dish was canted at an angle to induce the roots to grow downwards and against the paper. Dishes were kept at 25 C for 24 h. The seedlings were then carefully removed and the positions of the root tips marked. The filter papers which had been placed beneath the seedlings to absorb any exuded substances were air-dried and afterwards sprayed with 0.25% Ninhydrin in ethanol to detect exudation of amino acids, amides and amines (Pearson and Parkinson, 1961). To detect the sugars, the papers were sprayed rapidly and evenly with a mixture containing equal volumes of silver nitrate (N/10) and ammonia solution (5 N) and the paper dried in the oven at 105 C for 5-10 min. Regions of exudation were determined by comparing tlie growth and location of plant parts with the patterns of exudation exhibited on the paper. Collection of root exudates. Exudates were collected by the method of Odunfa (1977). Preparation of spore suspension. Conidia were obtained from 15-day-old PDA cultures growing on agar slants in an illuminated incubator. The agar tubes were half filled with sterile distilled water, shaken gently for 5 min with a Griffith wrist-action shaker and the conidial suspension filtered through a coarse cheese nylon. The filtrate was washed three times with distilled water by centrifuging and decanting the supernatant. The final spore suspension was made by adding sterile distilled water and making up the concentration to 5 X l Spore germination tests. One ml of the spore suspension of each fungus was added to 2 ml of root exudate in a universal bottle, placed in a small beaker and shaken at 20 rev/min on a Gyrorotary shaker at 26 ± 1 C in tbe dark. After an incubation period of 12, 24 or 48 h, percentage germination was determined by counting the germinated conidia on haemocytometer slides. A spore was considered germinated when its germ tube had attained a length equal to half the length of the macroconidium. Mycelial growth in exudate-supplemented liquid media. Preliminary experiments showed that no appreciable growth was obtained when fungi were inoculated into root exudates alone. Consequently a root exudate growth medium supplement with inorganic salts and some trace elements was developed. The trace elements stock solution included FeSO4 0.5 g, ZnS g, CUSO g, H3BO3 0.5 g, CaCls 0.5 g, MnCl2 0.5 g, M0O3O.O2 g, NaMo g, all dissolved in 1000 ml distilled water. The medium had the following composition: KH2PO g; MgS04. 7H2O 0.75 g; trace element solution 2 ml; root exudate solution 1 1; ph was adjusted to 5.5 with 5 N KOH. A ph of 5.5 was found to be optimal for the growth

3 Effects of root exudation on Fusarium 609 of the Fusarium test species. Distilled water was substituted for the root exudate in the control. Media were sterilized using a Pyrex millipore filtration apparatus. Twenty ml of each medium were dispensed into 100-ml conical flasks. Each was inoculated with the test Fusarium using a No. 2 cork borer and incubated in a growth room at 29 ± 1 C on a Lab-line Gyrorotary shaker set at a low speed of agitation. After 7 days the fungal mycelium from each flask was filtered onto a previously dried and weighed Whatman No. 1 filter paper (9 cm diam.), dried in an oven at 80 C for 24 h, cooled in a dessicator and weighed. RESULTS Sites of Exudation The exudation patterns are illustrated in Plates 1 and 2. Germinating seeds exuded detectable quantities of ninhydrin positive substances as early as 10 h after placing on moist filter papers. Larger quantity of amino acids were exuded by cowpea seeds. Seed exudation decreased with the development of the roots but ceased completely after 2 days. In seedlings, the root tips of primary and lateral roots of cowpea and also the root tips of adventitious roots of Sorghum were the major sources of exudation. The older parts of the roots also exuded detectable and significant quantities of organic compounds. Similar exudation patterns were obtained by developing with ammoniacal silver nitrate solution. Spore germination in exudates Germination of conidia was observed in the exudates of both species (Table 1). The rate of germination was highest in Fusarium solani II and lowest in F. oxysporum. The rate of Table 1. Percentage conidial germination of Fusarium species in root exudates of cowpea and sorghum at various times Fungal species Fusarium solani I F. solani II F. oxysporum F. semitectum Control (sterile distilled water) 12 h 24 h 48 h ±1 30± ±l 5± 12±2 Sorghum root exudate 12h 24 h 48 h ±2 23±2 42± ± ±7 100±8 100±4 12h *Germ tubes of germinating conidia were lysed. Figures represent the mean and standard deviations of three replicate experiments. Cowpea root exudate 24 h 98±4 100± ±7 48h 98±11* 100±7 93±8 100±9* gerpiination was very high in cowpea root exudates: this was followed except in one case by complete lysis of the cells. The rate of germination was slower in sorghum exudate but no lysis was noticed. Some germination was also recorded in the distuled water control. Growth of mycelia in exudates The mycelial dry weights in each of the media are shown in Table 2. Cowpea root-exudate medium was the only one which supported fairly good growth of the test fungi. In the medium, mycelial dry weight was found to be hi^est in F. semitectum and least in F. solani I. Sorghum root exudate medium did not increase mycelial growth appreciably above the control.

4 610 V. S. AYO ODUNFA Table 2. Growth of various isolates in media containing exudates of cowpea and sorghum Fungal species Fusarium solani I F. solani II F. oxysporum F. semitectum Basal medium + distilled water (control) 13±L5* 15±2.5 14± L8 Mycelial dry weights (mg/20 ml medium) Basal medium Significance Basal medium + of difference + sorghum exudate * 17±2.0 15±L from control n.s. cowpea exudate 24± ±2.0 Significance of difference from control P = 0.05 *Mean and standard error of three replicate determinations using one composite exudate from 200 seedlings. n.s. not significant. DISCUSSION Pearson and Parkinson (1961) and Schroth and Snyder (1961) found that exudation was in the zone behind the root tip. This is also true of exudation in cowpea and sorghum seedling roots. From this study, however, exudation was not confined to this zone alone but was observed in the older parts of the root although quantitatively less. This can be correlated with the results of Rovira (1969) and McDougall and Rovira (1970) that some diffusible materials were found along the whole length of the roots. Profuse exudation of ninhydrin-positive substances from the seed of cowpea and scanty exudation from sorghum seed was revealed. This is significant since there is evidence that seed exudation contributes significantly to the growth and nutritional level of both seedsurface fungi and spermatosphere organisms, including pathogens, thereby increasing disease development (Hayman, 1970; Baker and Cook, 1974; Short and Lacy, 1976). The phenomenally high percentage of seedling mortality in cowpea (Williams, 1975) might, in part, be explained by this abundant seed exudation. The results of conidial germination in distilled water and exudates demonstrate that there is no specific stimulation oi Fusarium conidial germination by the exudates of either species. Despite conflicting evidence it seems that some fungi like Verticillium albo-atmm (Schreiber and Green, 1962) and Sclerotium cepivorum (Coley Smith and King, 1970) require specific growth factors in the exudates while many, including Fusarium, are activated by a fairly broad range of unspecific nutrients. The lysis oi Fusarium germlings such as observed in the exudates of cowpea has been ascribed to high nutrient concentration, the kind of nitrogen in the substrate or some other unknown factors (Toussoun, 1970). Despite this, however, many pathogens still infect the root at the region of root elongation as described by Baker and Cook (1974). The growth of mycelium in root exudate is relevant to root colonization and pathogenesis for many soil-borne pathogens are supported by nutrition from root exudates (Snyder, 1970). The infrequent isolation of fusaria from soils (Thomas and Parkinson, 1967) and their predominance in the rhizosphere, rhizoplane, and geocarposphere of many plants suggest that they frequently respond to the presence of organic substrates from the root. The suitability of cowpea exudates for the mycelial growth oi Fusarium species may thus render cowpea susceptible to many root diseases. The suppression of mycelial growth in sorghum exudate indicates that its exudate contains some inhibitory substances, possibly hydrocyanic acid (Rangaswami and Balasubramanian, 1963) and tannins (Anderson, 1952), both of which have frequently been implicated in disease resistance.

5 Effects of root exudation on Fusarium 611 REFERENCES ANDERSON, A. M. (1952). Cause of reddening of roots and effect of fungi on sorghum seedlings. Proc. Ass. Of fie. Seed Anal. 42, 117. BAKER, K. F. & COOK, R. J. (1974). Biological Control of Plant Pathogens. W.H. Freeman and Company, San Francisco. CO LEY SMITH, J. R. & KING, J. E. (1970). Response of resting structures of root infecting fungi to host exudates: an example of specificity In: Root Diseases and Soil-borne Pathogens (Ed. by T. A. Toussoun, R. V. Bega & P. Nelson), pp University of California Press, Berkeley. HAYMAN, D. S. (1970). The influence of cotton seed exudates on seedling infection by Rhizoctonia solani. In: Root Diseases and Soil-borne Pathogens (Ed. by T. A. Toussoun, R. V. Bega & P. Nelson), pp University of California Press, Berkeley. MCDOUGALL, B. M. & ROVIRA, A. D. (1970). Sites of '''C-labeUed compound from wheat roots. New Phytol, 69,999. ODUNFA, V. S. A. (1975). Studies on the rhizosphere microorganisms of cowpea and guinea corn. Ph.D. thesis. University of Ibadan, Nigeria. ODUNFA, V. S. A. (1977). Free amino acids in the seed and root exudates in relation to the nitrogen requirements of rhizosphere soil Fusaria. Can. J. Bot, {in press) PEARSON, R. & PARKINSON, D. (1961). The sites of excretion of ninhydrin positive substances by broad bean seedlings. Plant and Soil, 13, 391. RANGASWAMI. G. & BALASUBRAMANIAN, A. (1963). Release of hydrocyanic acid by sorghum roots and its influence on the rhizosphere microfiora and plant pathogenic fungi. Indian J. expt. Biol. 1, 215. ROVIRA, A. D. (1969). Diffusion of carbon compounds away from wheat roots. Aust. J. Biol. Sci. 22,1287. SCHREIBER, L. R. & GREEN, R. J. (1962). The effect of root exudates on germination of conidia and microsclerotia of Verticillium alho-atrum inhibited by soil fungistatic ptjnciple, Phytopathology, 53, 260. SCHROTH, M. N. & HILDEBRAND. D. C. (1964). Influence of plant exudates on root-infecting fungi. Ann. Rev. Phytopathol, 2, 101. SCHROTH. M. N. & SNYDER. W. C. (1961). Effect of host exudates on chlamydospore germination of the bean rot fungus Fusarium solani f. phaseoli. Phytopathology, 51, 389. SHORT. G. E. & LACY, M. L. (1976). Carbohydrate exudation from pea seeds: Effect of cultivar, seed age, seed colour and tempeiature. Phytopathology, 66, 182. SNYDER. W. C. (1970). Recent advances in the study of ecology of soil-borne plant pathogens In: Root Diseases and Soil-borne Pathogens (Ed. by T. A. Tousson, R. V. Bega & P. E. Nelson), pp University of California Press, Berkeley. TOUSSOUN, T. A. (1970). Nutrition and pathogenesis of Fusarium solani f.sp. phaseoli. In: Root Diseases and Soil-borne Pathogens (Ed. by T. A. Tousson, R. V. Bega & P. E. Nelson), pp University of California Press. Berkeley. WILLIAMS. R. J. (1975). Control of cowpea seedling mortality in southern mgeria. Plant Dis, Reptr, 59, 245. EXPLANATION OF PLATES PLATE 1 Exudation patterns of cowpea seed and seedlings on Whatman No. 3 filter paper developed with ninhydrin. No. 1. Seed exudation from cowpea seeds after 24 h. The black patches, a, are areas with ninhydrln-positive substances: b. position of emerging radicle. No. 2. Seed exudation 3 days after plating; a. outline of original position of seed. No. 3. Root exudation in 4-day-old cowpea seedlings. No. 4. Root exudation pattern in 6-day-old cowpea seedlings; a, position of seed remain. PLATE 2 Exudation pattern of Sorghum 'seed' and seedlings on Whatman No. 3 filter paper developed with ninhydrin. No. 5. Seed exudation from Sorghum seeds after 24 h of plating; a, position of Sorghum 'seeds'.

6 612 V. S. AYO ODUNFA No. 6. Root exudation 3 days after plating; a, position of Sorghum 'seeds', b, portion of the root with little or no exudation, c, root tip of primary root with exudation. No. 7. Root exudation pattern of a 4-day-old Sorghum seedling. No. 8. Root exudation pattern of a 6-day-old Sorghum seedling; a, position of 'seed' remain; b, root tip of adventitious roots (exudation is profuse here); c, older portion of adventitious root with moderate exudation.

7 THE NEW PHYTOLOGIST. 80, 3 PLATE 1 ;. " it. 2 3 V, S. AYO ODVNFA EFFECTS OF ROOT EXUDATION ON FUSARIUM {facing p. 612)

8 THE NEW PHYTOLOGIST, 80, 3 PLATE 2 b c 5 «r b c> C -a 8 b V. S. AYO ODUNFA OF ROOT EXUDATION ON FUSARIUM

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