Phytopath. 2., 105, 351—359 (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

By

F. M. SHAO and M. N. CHRISTIANSEN

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 (ROVIRA

1956, 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 (KATZNELSON

et al. 1954), low light intensity (ROVIRA 1956), oxygen deficiency (CHRISTIAN-

SEN et al. 1970, GRINYOVA 1961), low temperature (ROVIRA 1956, HAYMAN

1969), 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

352 SHAO and CHRISTIANSEN

(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 and

HiLDi-BRAND 1964 and SCHULTZ and BATEMAN 1969).Tn this study, we examined the relationship of cotton seedling radicle

exudation at 10 C and susceptibility to Verticillium wilt and Rhizoctoniaseedling damping-off and root rot of cotton.

Materials and Methods

Collection of exudates

Acid-delinted cotton seed Acala 4852 were treated with 1.3/ig/kg Ceresan L. (2—8%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 exudates

The exudates were qualitatively analysed for amino acids and sugars by thin-layerdiromatography (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 (SNELL

et al. 1961). Data from five replications were subjected to statistical analysis.

Testing for susceptibility

After collecting the exudates the seedlings were subsequently planted directly into 1 kgof 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.

Results

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 3—4 23

SHAO and CHKISTIANSEN

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 stein

Leucme

Methionine

PhenylalanineSerineTryptophanValineUnknown

0

—

—

+————

———

Days at 10 °C

3

+

++++

+1

5

++

+——+++

1

+ = 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

30

OLJLUCO

COLJ

XLU

20

0 0 AMINO ACIOS

O O SUGARS

I

-•DISEASE SEVERITY

XLUQ

crUJUJ(0

UJCO

UJCOo

DAYS AT 10 C

Fig. 2. Relationship between seedling radicle exudation at 10 °Cand 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)

0

2345

02345

0

2345

0

2

345

Pathogen

Uninoculated

R. solani

V. alho-atrum (SS^)

V. alho-atrum (T-l)

Disease severityindex''")

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

0-0 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 b

3.46 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 class

number ot inoculated plants

"•''•) In each vertical column, means followed with the same letter are not significantlydifferent according to Duncan's Multiple Range Test.

336 SHAO and CHRISTIANSEN

DAUX \OV the overall disease severity, top dry weight and total aminoacicl-s And sugars oxud.itcs is summarized in Table 3. The relationship of diseasescvcrit\' and top dry weight to amino acid and sugars exudation is shown inTable 4 and in Figures 2 and 3. These data indicate that both disease severity

30

20oUJaiw

LU

Q3XLU

10

I I IAMINO ACIDS

O o SUGARS

• — • TOP DRY WT

I I

o

I

LU

O

D A Y S A T I O C

Fig. 3. Relationship between seedling radicle exudation at 10 "̂ C and field (top dry weight)of cotton

Tahle 3Effect of chilling at 10 '̂ C on disease severity, top dry weight and exudation

by cotton {Gossypium hirsutum L.)

Chilling time(days at 10 ̂ 'C)

0

2

34

5

Disease severityindex'-')

1.40 d'̂ -̂ '-)1.43 dc1.70 be1.85 ab2.10 a

Top dry weight(g)

2.19 a2.00 ab1.80 be1.56 cd1.45 d

Exudates (//g/seedling)

amino acids sugars

8.60 b 9.12 b13.60 a 10.44 b14.60 a 10.44 b14.80 a 14.20 ab15.40 a 17.36 a

See Table 2, footnote ''•').See Table 2, footnote '''"'').

Cotton Seedling Radicle Exudates in Relation to Verticillium Wilt 357

and top dry weight are correlated with amino acids and sugars exudation.Disease severity increased significantly with increase in amino acid and sugarsexudation while top dry weight decreased also significantly.

Tahie 4Correlation of disease severity and top dry weight to amino acid and sugars exudation

DiseaseTop dry

severityweight

index

Correlation

amino acids

0.7385 NS—0.8526 :̂-

coefficients (r)''')

sugars

0 . 9 4 2 3 =•"••

—0.9241 =̂-

'•') Double asterisk ("'"'•) indicate correlation coefficient significant at P = 0.01. Singleasterisk ('•") indicate correlation coefficient significant at P = 0.05. NS indicate correlationcoefficient not significant at P 0.05.

Discussion

The cultivar of cotton used in this study is known to be tolerant toVerticillium wilt (WILHELM et al. 1974). BOOTH (1969, 1974) found thatVerticillium wilt susceptible cultivars of cotton produced greater amounts ofL-alanine (2 mM) increased Verticillium growth 320% (dry weight basis), butwhen 2.0,ng/ml choline was added to the above growth was reduced to 41 %of control cultures. Polygalacturonase activity was also affected by the treat-ments, 2 mM L-alanine, 17.2 units/mg dry weight of growth vs. 10.4 units forcontrol cultures and 7.4 units for L-alanine + choline. Choline alone had littleeffect on growth or polygalacturonase production. When L-alanine was intro-duced into plants of the tolerant cultivar before inoculation, it restored toler-ance (BOOTH 1974, SINGH et al. 1971). Our findings that the Verticillinm wilttolerant cotton used in this study was significantly more susceptible to thedisease after exposure to cold stress at 10''C for 3 and 5 days and thatL-alanine formed the highest concentration of an individual amino acid inthese exudates seem to correspond with the findings of the previous workers.

Summary

Seedlings of the Verticillium tolerant cotton cultivar Acala 4852 weresubjected to chilling at 10 °C. Radicle exudates were taken after 2—5 daysand the chilled plants were examined for susceptibility to Verticillium wiltand Rhizoctonia root rot. Exudates were analysed for amino acids and sugarsby fluorometric and colorimetric procedures. Disease severity increased signifi-cantly with increase of amino acid and sugars exudation, while top dry weightdecreased also significantly. From the qualitative analysis of exudates L-alanine seemed to form the largest concentration of an individual amino acidin both the 3 and 5 days exudates; in the unchilled control L-alanine was notidentified. Our findings that VerticilUum wilt tolerant cotton was more sus-

358 SHAO and CHKISTIANSEN

cptiblc to tho disc.isc alter exposure to cold stress seem to correspond with theiiidini;s ol UiL' previous workers.

Zusammenfassung

Kclmpflanzenwurzelexsudate der BaumwoUein Relation zur Anfalligkeit gegenuber Verticiliiam-

und Rhizoctonia-

l\LMniptlan/en der Verticillinm-ioleranien Baumwollsorte Acala 4852\\'urden einer Temperatur von 10 "C ausgesetzt. Wurzelexsudate wurden nach/wei his funf Tagen gesammelt und die mit Kalte behandelten Pflanzen wur-den auf ihre Anfalligkeit fiir Verticillii4m-\(/e\ke und Rhizoctonia-Wurzeli'iulQgcpriift. Die Exsudate wurden fluorometrisch und kolorimetrisch auf ihrenGehalt an Aminostickstoffverbindungen und reduzierenden Zuckern analysiert.Zwischen dem Krankheitsbefall und der Exsudatmeage wurde eine positiveund zwischen dem Trockengewicht der Pflanzen und der Exsudatmenge einenegative Korrelation festgestellt. In den wahrend drei und fiinf Tagen bei10 C gehaltenen Pflanzen wurde L-Alanin in der hochsten Konzentrationemer einzelnen Ammosaure ldentifiziert; m nicht kaltebehandelten Pflanzenwurde L-Alanin nicht nachgewiesen. Diese Ergebnisse konnen frlihere Befundebestatigen, dafi Welke-tolerante Baumwollsorten bei tiefer Temperatur an-fallig sind.

We would like to thank A. A. BFLL and N. R. O 'NEIL for respectively providing theVcrticilliuyn and Rhi'zoctonia isotopes u^ed in the study. Our appreciations are due to C. BARE

tor assisiancc in the analysis of the exudates and H. E. HEGGESTAD, N . B. MANDAVA, R.

TAYLORSCN and R. K. HONX tLL for reviewing the manuscript.

Literature

BARLO\̂ ^ R., Jr., 1970: Critical requirements for genetic expression of Verticilliitm wilt toler-ance in Acala cotton. Phytopathology 60, 559—560.

BELL, A. A., and J. T. PRESLEY, 1969: Temperature effects on resistance and phytoalexin syn-thesis in cotton (Gossypium sp.) inoculated with Verticillium albo-atrum. Phytopatho-logy 59, 1141 — 1146.

BEKriv, S. Z., and C. A. THOMAS, 1961: Influence of soil temperature, isolates and method ofinoculation on resistance to Verticillium wilt. Phytopathology 51, 169—174.

BOOTH, J. A., 1969: Gossypium hirsutnm tolerance to Verticillium albo-atrum infection. 1.Amino acid exudation from aseptic roots of tolerant and susceptible cotton. Phyto-pathology 59, 43—46.

, 1974; Effect of cotton root exudate constituents on growth and pectolytic enzyme pro-duction Verticillium albo-atrum. Canad. J. Bot. 52, 2219—2224.

BuGBEE, W. M., and J. T. PRESLEY, 1967: A rapid inoculation technique to evaluate theresistance of cotton to Verticillium albo-atrum. Phytopathology 47, 1264.

CHRISTIANSEN, M . N . , 1963: Influence of chilling upon seedling development of cotton. PlantPhysiol. 38, 520—522.

, 1964: Influence of chilling upon subsequent growth and morphology of cotton seedlings.Crop Sci. 4, 584—586.

, R. H. CARNS, and D. J. STYLER, 1970: Stimulation of solute loss from radicles ofGossypium hirsutum^ L. by chilling, anaerobiosis and low pH. Plant Physiol. 46, 53—56.

Cotton Seedling Radicle Exudates in Relation to Verticillium Wilt 359

, and R. O. THOMAS, 1969: Season-long cffcct.s of chillinj^ treatments applied to germi-nating cotton seeds. Crop Sci. 9, 672—673.

GRINYOVA, C . M., 1961: Excretion of substances by plant roots during brief anaerobiosis.Fiziol. Rast. 8, 686—691.

HALISKY, P. M., R. H. GARBER, and W. C. SCHNATHORST, 1959: Influence of soil temperatureon Verticillium hydromycosis of cotton in California. Plant Dis. Reptr. 43, 584—588.

HAYMAN, D . S., 1969: The influence of temperature on the exudation of nutrients from cottonseeds and on pre-emergence damping-off by Rhizoctonia solani. Canad. ). Bot. 47,1663—1669.

KATZNELSON, H . J., ROVATT, and T. B. M. PAYNE, 1954: Liberation of amino acids by plantroots in relation ro desiccation. Nature 174, 1110—1111.

MALAJCZUK, N . , and A. J. MCCOOMB, 1977: Root exudates from Eucalyptus calophylla R.Br.and Eucalyptus marginata Donn. ex Sm. seedlings and their effect on Phytophthoracinnamomi Rands. Austral. J. Bot. 25, 501—514.

MCCARTER, T. M., and R. W. RONCARDORI, 1971: Influence of low temperature during cottonseed germination on growth and disease susceptibility. Phytopathology 61, 1426—1429.

MINTON, E. B., and J. R. CUPSON, 1978: Effects of controlled night temperatures on Verti-cillium wilt of field grown cotton. Proc. 1978 Beltwide Cotton Prod. Res. Conf., p. 26.

ROVIRA, A. A., 1956: A study of the development of the surface microflora during the initialstages of plant growth. J. Appl. Bact. 19, 72—79.

, 1965: Plant root exudates and their influence upon soil microorganisms. In: BAKER

and SNYDER (eds.). The Ecology of Soilborne Plant Pathogens, 170—186. Univ. Cali-fornia Press, Berkeley, Calif.

, 1969: Plant root exudates. Bot. Rev. 35, 35—57.SCHNATHORST, W . C , 1964: A fungal complex associated with sudden wilt syndrome in

California cotton. Plant Dis. Reptr. 48, 80—92., and D. E. MATHRE, 1966: Host range and differentiation of a severe form of Verti-

cillium albo-atrum in cotton. Phytopathology 56, 1155—1161.SCHROTH, M . N . , and R. J. COOK, 1964: Seed exudation and its influence on pre-emergence

damping-off on bean. Phytopathology 54, 670—673., and D. C. HILDEBRAND, 1964: Influence of plant exudates on root infecting fungi.

Ann. Rev. Phytopath. 2, 101—132.SCHULTZ, F . A., and D. F. BATEMAN, 1969: Temperature response to seeds during early phases

of germination and its relation to injury by Rhizoctonia solani. Phytopathology 59,352—355.

SINGH, D., E. A. BRINKERHOLFF, and G. CUINN, 1971: Effect of alanine on development ofVerticillium wilt in cotton cukivars with different levels of resistance. Phytopathology61,881—882.

SMITH, W . H . , 1976: Tree root exudates and the forest soil ecosystem: exudate chemistry,biological significance and alteration by stress. In: J. MARSHALL (ed.), The Below-ground Ecosystem: A Synthesis of Plant Associated Processes. Dowden, Hutchinson andRoss, Stroudsbury P.A.

SNELL, F . D., C . T . SNELL, and C. A. SNELL, 196): Colorimetric Methods of Analysis,165—166. D. Van Nostrand and Co., New York.

STAHL, E., H . R . BOLLINGER, M . BRENNER, G. GANSHIRT, H . R . MANGOLD, H . SFILLER, and

D. WALDI, 1965: Thin-layer Chromatography. A Laboratory Handbook, 553 pp.Academic Press., Inc., Publ., New York and London.

UDENFRIEND, S., S. STEIN, S. BOHLEN, and W. DAIRMAN, 1972: A new fluorometric procedurefor assay of amino acids. Peptides and proteins in the Pincomole Range. Third Amer.Peptide Symp., Boston, June 1972.

WILHELM, S., J. E. SAGEN, and H. TIETZ, 1974: Gossypium hirsntu^yt subsp. Mexican var.nervosum, Lenningrad strain. A source of resistance to Verticillium wilt. Phytopatho-logy 64, 931—939.

Authors' address: Plant Stress Laboratory, Plant Physiology Institute. Belrsville Agri-cultural Research Center, Beltsville, Maryland 20705 (U.S.A.).