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  • Phytopath. 2., 105, 351359 (1982)(c) 1982 Verlag Paul Parey, Berlin und HamburgISSN 0031-9481 / InterCode: PHYZA3

    Cooperative Investigations of Agricultural Research,U.S. Department of Agriculture, Beltsville, Maryland 20705,

    and Division of Plant and Soil Sciences,West Virginia University^ Morgantown, WV 26506

    Cotton Seedling Radicle Exudatesin Relation to Susceptibility to Verticillium Wilt

    and Rhizoctonia Root Rot



    With 3 figures

    Received October 22, 1981

    Plant root exudates have received considerable attention because theyhave a great influence on populations of soil microorganisms generally (ROVIRA1956, 1965, 1969), and particularly on soilborne plant pathogens and plantroot symbionts (ROVIRA 1956, SCHROTH and COOK 1964, SCHROTH and HILDE-BRAND 1964, SMITH 1976).

    Factors which promote root exudation include drought (KATZNELSONet al. 1954), low light intensity (ROVIRA 1956), oxygen deficiency (CHRISTIAN-SEN et al. 1970, GRINYOVA 1961), low temperature (ROVIRA 1956, HAYMAN1969), and low pH (ROVIRA 1956). Low temperatures have further been shownto influence growth, morphological development, susceptibility to diseases andyields of cotton (CHRISTIANSEN 1963, 1964, HAYMAN 1969, MCCARTER andRONCARDORI 1971) .

    Verticillium wilt of cotton {Gossypium hirsutum L.) caused by the micro-sclerotial form of Verticillium albo-atrum Reinke and Berth (also known asV. dahliae Klebahn), SCHNATHORST (1964) has been found to be most severe attemperatures 10'^C to 27 C (MINTON and CUPSON 1978). An optimum daytemperature of 24.5 ^C was found necessary to separate genetically differentstrains of cotton when inoculated with a mild strain of V. albo-atrum (SS-4)(BARROW 1970). BELL and PRESLEY (1969) observed that a tolerant variety ofcotton was classified as susceptible at 25 ' C, tolerant at 27 ^C and resistantat 29 C when it was inoculated with a defohating strain of V. albo-atrum

    U.S. Copyright Clearance Center Code Sutement: 0 0 3 1 - 9 4 8 1 / 8 2 / 0 5 0 3 - 0 3 5 1 ^ 0 2 . 5 0 / 0


    (T-l). In pui-L- culiurc, the growth of V. albo-atrum was found to be equallyretarded at 10 C and 30' C (BEKRY and THOMAS 1961); but HALISKY et ai(1950) reported an optimum temperature of 24 "C.

    Rhiy.octonia seedling damping-off caused by Rhizoctonia solani Kuhn hasalso been reported to be more serious at low temperatures (HAYMAN 1969MCCARTER and RONCARDORI 1971, SCHROTH and COOK 1964, SCHROTH andHiLDi-BRAND 1964 and SCHULTZ and BATEMAN 1969).

    Tn this study, we examined the relationship of cotton seedling radicleexudation at 10 C and susceptibility to Verticillium wilt and Rhizoctoniaseedling damping-off and root rot of cotton.

    Materials and MethodsCollection of exudates

    Acid-delinted cotton seed Acala 4852 were treated with 1.3/ig/kg Ceresan L. (28%methyl-mercury 2,3-dihydroxypropyl-mercaptide and 0.62% methyl-mercury acetate) thengerminated in paper towels at 30 "C for 24 to 30 hours. Seedlings with radicles about 2 cmlong wore selected and transferred to stainless steel racks in petri dishes so that 1 cm of theseedling axis was immersed in sterile distilled water (50 ml). Each rack contained 10 seedlings;there were six racks per treatment (six replications). These were subjected to chilling at 10'^ Cfor 2, 3, 4 and 5 days. A control treatment consisted of six racks of 10 seedlings eadi, sub-jected to 30 "C for one day. After completion of each treatment, the liquid Ln each dish wasfiltered and transferred to a freeze-drying flask and lyophiiized. The dry residue was dissolvedin 2 ml of water, filtered and assayed for amino acids and sugars.

    Analysis of the root exudatesThe exudates were qualitatively analysed for amino acids and sugars by thin-layer

    diromatography (TLC) method (STAHL ct al. 1965). Solvents used included n-butyanol-aceticacid-water (60 + 20 + 20) and t-butanol-methyl ethyl ketone-formic acid-water (40 + 30 + 15+ 15) for amino acids and sugars respectively. Exudate samples of 50//I were used and com-pared with 5 //I of some known standard amino acids and sugars for identification purposes.Cellulose plates were used for the determination of amino acids and silica gel plares for sugars-These were sprayed with ninhydrin and aniline phthalate respectively.

    Quanntative determination of amino acids in the exudates was achieved by fluororaetricprocedures (UDENFRIED et al. 1972) and sugars by the colorimetric method of analysis (SNELLet al. 1961). Data from five replications were subjected to statistical analysis.

    Testing for susceptibilityAfter collecting the exudates the seedlings were subsequently planted directly into 1 kg

    of steam sterilized soil in 10 cm pots. The soil was fertilized with 100, 103, and 137///kgNjPjKj, respectively from solutions of NH^NO_^ and K^HPO^ (5.5 ml). Three seedlings wereplanted per pot and the pots were kept in the greenhouse at about 30 C for two weeks. Thepots were then moved to a growth diamber maintained at 25 C day temperature and lightedwith incandescent light to give 16 hours of light. They were inoculated with V-albo-atrumisolates T-1, and SS^, a severe defoliating strain and a mild non-defoliating strain, respectively(SCHNATHORST and MATHRE 1966). A mixture of two R. solani strains isolated from Poa andLolium spp., both belonging to anastomosis group four was used. Verticillmm inoculation wasachieved through the needle puncture technique (BUGBEE and PRESLEY 1967) while Rhizoctoniainoculation was performed using infested oat seed inoculum placed around the hypocotyl ofeach plant and covered with sterile soil.

  • Cotton Seedling Radicle Exudates in Relarion to Vcrticillinm Wilt 353

    The plants were scored for disease severity on a scale of 0 to 4 where 0 -^ healthy plantand 4 = dead plant, three weeks after inoculation.

    The experimental design in the chamber was a completely randomized block with fivereplicates.


    The amino acids in the exudates were identified as shown in Table 1.Only aspartic acid was detectable in trace amounts in the unchilled controlwhile ten amino acids were identified in the exudates from seedlings chilled at10'^C for 3 and 5 days. From the qualitative analysis of exudates (Fig. 1),L-alanine seemed to form the largest concentration of an individual aminoacid in both the 3 and 5 days exudates.

    Identification of the sugars also revealed no detectable amounts in theunchilled control and only traces in the exudates from plants chilled at 10''C

    0 3 5 A C D F L M N S V

    Fig. 1. Qualitative analysis of amino acids in exudates from seedling chilled at 10-'C for 0, 3and 5 days by thin layer chromatography. A = L-alanine, C = cysteine, D = asparticacid, F = phenylalanine, L = leucine, M = methionine, N = asparagine, S =- serine,

    V =- valine, W = tryptophane

    Phytopath. Z., Bd. 105, Heft 34 23


    Tahle Iacids ci^nsiitucnts ol exudates i rom cotton seedlings chilled at 10

    from 0, 3 and 5 days

    Ammo .UKIS

    L-alanineAsparagineA-spartic acidCy steinLeucmeMethioninePhenylalanineSerineTryptophanValineUnknown



    Days at 10 C3









    + = Amino acid spotted on T L C plate from 50 / / I sample,identified on T L C pla te from 50 //I sample.

    = Amino acid not













    DAYS AT 10 CFig. 2. Relationship between seedling radicle exudation at 10 C

    and susceptibility to Verticillium wilt and Rhizoctonia root rot diseases of cotton

  • Cotton Seedling Radicle Exudates in Relation to Verticillium Wilt 355

    for 5 days. Glucose was the only sugar identified in the exudates from plantschilled at lO^'Cfor 3 days.

    The results of the effect of chilling at 10 ^C on disease severity caused byV. alho-atrum and R, solani are summarized in Table 2. Disease severitycaused by strain T-l was significantly higher among seedlings chilled at 10 "^ '^Cfor three to five days, compared to those chilled for 0 and 2 days. Top dryweight was significantly reduced after five days at 10'^'C. This may be due tothe severe nature of this strain because it reduced yield significantly comparedto the other pathogens. With the mild strain (SS4), disease severity was sig-nificantly increased only after five days of chilling at 10 "^'C, but top dryweight reduction was significant after three and five days. When the root rotpathogen R. solani was used, disease severity was significantly increased withseedlings chilled at 10 '^C for four to five days and yield reduction after threeto five days.

    Table 2Effect of chilling at 10 '"C on disease severity and top dry weight

    of cotton {Gossypium hirsutum L.)

    Chilling time(days at 10 ^C)







    R. solani

    V. alho-atrum (SS^)

    V. alho-atrum (T-l)

    Disease severityindex''")

    0.0 g='="-)0.0 g0.0 g0-0 g0.0 g

    1.52 f1.52 f1.80 ef2.40 c2.58 c

    1.58 ef1.80 ef1.90 ef1.96 de2.36 c

    2.26 cd2.54 c3.04 b3.04 b3.46 a

    Top dry weight(g)

    3.05 a2.76 ab2.67 abc2.54 bed2.35 cde

    2.54 bed2.54 bed1.95 efg1.90 fg1.60 gh

    2.05 ef2.02 efg1.53 gh1.62 gh1.34 hi

    1.16 ij1.05 ij0.85 jk0.83 jk0.51 k

    '') Disease severity based on a scale of 0 to 4 where 0 ^ healthy plant and 4 = deadplant. Disease severity index was calculated from the equation;

    number of infected plants X severity classnumber ot inoculated plants

    "'') In each vertical column, means followed with the same letter are not significantlydifferent according to Duncan's Mul


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