Effect of the combination of seed priming and Trichoderma treatment on incidence of damping-off agents.

l32l

Med. Fac. l:ndbouww. Univ' Gent,58/3b' i993

EFFECT OF THE COMBINATION OF SEED PRIMING AND Trichoderma TREATMENT ON INCIDENCE OF

DAMPING-OFF AGENTS

B. JACQMIN, A.M. COTES, P. LEPOIVRE & J. SEMAL Faculté des Sciences Agronomiques de Gembloux

Laboratoire de Pathologie Végétale

Passage des Déportés 2, 8-5030 Gembloux' Belgique

8UI.iMÀRY

cucumber and Tettuce seeds elere osmoprimed (1or 48 and 72

h, r'espectiveTy) in a mixture of poTyethyTen-e g!yc27 soTution

una vàrniculité. one day beloie the end of the prining

proced.ure, the process vas intertupted in order to dip seeds

7à, 70 iin in' a spore suspensron of Trichoderna koningii .

^ft";

priming,,""â" were pTanted in sand infested with pvthiun sp. -Bean seeds vere -primed in water aqar for 24 h,

l'ré"iàà ,itn Trichoderma jus{ before prining, and tested in

sand inlested with Pythiun or with R. sol-ani. The percentage

of emergence was obselved after I'o days of incubation'

Treatment of unprined seeds with Trichodetma sTighxTy

increased stands, rèTative to seeds unprimed and untreated with Trichoderma. Prininq seeds consistentTy resulted in more rapidTy emergence with stands ranging. lrom 622 to 882

yaZcorâing to the crop-pathogen combinations). as compared to iOZ - Ze{ for the unprlned ànd Trichoderma-untreated control-.

Conbining Trichodermâ treatnent and seed prining increased

pTant siand.s (emergences ranging lron 882 to yOOZ) _{| relative}

io untreated prinea control, except for the.Pythium-lettuce and Rhizoctoiia-bean combinations, for wbich Trichoderma

treatment did not provide any additionaT protection xo seed

prining in terms of getmination percentage.

- _{prlmed Trichoderna-treated cucumber and bean seeds tlere} treated etith lungicide sumico in ordet to ki71 Trichoderma,

and were then planted in sand infested with Pythiun ' we

observed that killing Trichoderna after prining procedure did

not nodify the protection obtained. INlRODUCTION

The use of biological control agents is potentially interesting to control soilborne plant diseases _' Seed treatrnents is an attractive delivery systen for fungal or bacteriat bi-oprotectants, and in this respect, the use of Trichoderma Àpp. as biol-ogical control agents is

1322

somewhat _{less effective and nore variable than}

chenicar.

pesticides. ?hus, _{.developping seed treatnent p.àtéa.r.", that}

enhance _{and stabirize lhe -efficacy of uioiogital control}

agents of plant pathogens _are needed

One of the most_ pr.omising physiological rnethods to enhance

the efficacy of biological agLnti is sied pr:.minj-1e1, in wich

control-1ed hydration inltiates ti

without raaiJre Lrnersence(z). o=*ontir*li!'Tà="1

"rl

"t";Ï:::;il;

to

in an

::T:!i"

slysor

",

sdrLS. r'ne osmorrc _{potentiaL 0f the solution regurates the}

ânount of water uptake by the seed, enabling tne -:.',.ùiutio' _ot

the germination _{processlé, 10, 11): solid *itii" p.îri"q (sMp)}

have. Jceen

-deve)-opped as 'a.t âlt"r.rative to -p;ïri;;" seeds in

osmotic _{solutions. In this procedure, seeds are rnixed with}

a

filçfy ground tignite or coa.l_ _{substance, anâ sufficient}

additionar water is added to achieve tne approp.iut" rnoisture

potential f or priming; the _{rni.xture is thË;} - _i'"""r.l"a

f or a

given time at constanÉ temperature (7).

The purpose of our work was _{to'evaruate the integration of}

the priming procedures _{and seed treatnents with rrichoderma,} on the incidence of damping_off fungi, eyiniuÀ' _=p. and

Rhizoctonia so-?.ani.

I.IÀTERIÀÈs AND IIIETEOD8

Seeds and nicroorqani-sms

seeds used in ttri-s study were cucurnber (cucumis _sativus,

var. Délicatesse), bean. (phàseoTus vuTgaris) ,rui. er.1,ra"; , and lettuce (Lactuca _{sativa, var.} paresseuse).'

À strain of Trichoderna koningii Rifai oÉtained from _{(MicrobioJ-ogical Research ce-nter, Andes university} crMrc

of Colonbia) was previously selected for its protective properties against pythiun sp. and R. solaÀi (unpublished

results) , \,rhen _{incorpôrated in the soil and as sJea coati-ng.}

strains of pythiun sp. and R. soTani r,rere isoralea in our ' l-aboratory from sugar beet with brack reg ..rà bean with

damping-off symptons.

Substrate

À11 experiments were perforned _in sand sieved through a 4

mn mesh _{screen and sterilized at 15ooc for 6 h, sowirig was} performed _{in plastic pots containing 32o g of sand in which}

s

bean seeds,5 cucurnbèr seeds or 10 lettuce seeds had been

planted.

Trichoderma seed treatrnents and priminq procedures

- . T. koningii was grown on malt extract agar (Difco) at 25oC

9urilg 7 days, under _a.16 h photoperiod of -fluorescent }ight.

conidia were harvested by scràping- _{the surface of the colonies}

a spore suspension of 107 spores per millj-Iiter. Unprimed

=eea= _(control).were d-ipped for l-0 rnin in this suspension, or in water

' _{eriming of cucunber and lettuee} seeds was performed by

rnaintaining thenr in Petri dishes (185 mm diameter) containing

îô-;-;i vermicurire rnoistened with 150 mt_ of pEG sotution ,3O.2 _{g/ 100m1) ( L5, 16) , under a layer of} Whatman No 1 f j-lter p.p"r. -éucumber and lettuce seeds were then incubated for 48 h à"â zz h, respectively, at 25"c under a 16 h photoperiod-of ii,.rot.=..tlt 1iqht. one day bef ore the end of the priming frocedure, the treatment wàs interrupted for 10 min in order t" imrnerse seeds in a spore suspension of T. koningii as described above (or in water for control) and was then

resumed. Bean _{=".à= were first} treated with T. koningii as

described above, and then prirned for 24 h in Petri dishes (85

mrn) contaj-ning water agar (22) in a growth chamber'

on primin! completion, seeds were rj-nsed with tap water for 20 sec -before beinq submitted to drying for 48-72 h at 25oc' Seeds were stored then in Petri dishes at 4oc'

InordertokillTrichodetmaafterprirning,seedswere

aippàa for 30 min in 4ppm (a.i) of the fungicide Surnico (252

diéLhofencarbe and iiZ carbendazime) (4), or in water

(control), and then dried.

Colonj,zation of bean seeds by ?rjchoderma

col_onization of seeds by Trichoderma was evaluated before

and after the drying proceàure. Three replicates of 2 g of seeds were washeâ i"i one minute with O.O52 Tween 20 in sterile distilled water, suspended in 18 m1 of steril-e distilled water and triturated i-n a nortar. Serial dilutions were prepared, and 1oo 1tI aliquots of each dilution were

pl-ated ônto o.1U sodium desoxycholate PDA, followed by

incubation for 5 days at 25oC under a fluorescent light with

16 h photoperiod ResultanL Trichoderma counts were expressed

a= coiony-iorning units (cfu) per gram of seed'

Gfowth chamber exPeriments

Pythiun was grown on nutrient mediun containing

Verrnitulite, V8 juice as additive, and water (?o g/ 24 nl/ eô*i r"rp"clivefyl. This mediurn was autocl-aved for 20 min at

!2OoC, ana was inoculated with 4 discs (5mm) of 7 day-o1d

pythiin culture grown j-n Corn meal agar. (CM, Difco) . R.so-lanj was grown on a nutrj-ent medium containi-ng 2O g of Vermi-culite

u"a do nL of liquid nedium yDB (19), autoclaved for 20 min at

iio'c, and inoiulated with 4 discs (5mm) of 7 day-old

Rhizoctonia culture grown in MEA. cultures were incubated for

7 days j,n the growth chamber as described above'

The sand was inoculated with Pythiun culture (0'012 _| O'IZ

or 3-OZ w/tl) for cucunber, bean or lettuce experiments

respectively, or with R. soTani (2'5e" W/W) for bean experirnents.

Prinedorunprj.medseeds(eithertreatedornotwithî'

koningii) w"r" _=oiun in pots fil1ed with infested or uninfested

Studies were conducted in a growth chamber aX 22 t L oc with à pt otoperioa of t 6 h li-ght. Sand moisture holding

capaci-ty'was ira1ntained at 6O?. Stand daily counts started 1

a"i, af€er planting and continued. for 10 days. Counts were

exiressed aÀ percentages of germination'

There were s reflicateé of each treatment arranged in a

randomized complete tlock design, on growth chamber benches'

The study was rePeated twice.

REgUIJTB

seedfinq assav with PYtàitll4

EmergenceofunprinredorTrichoderma-untreatedseedsin eythiun-iÂfested sand were 26?, 16? and 26% in cucumber, bean

and Iettuce, respecti-ve1y (Fi-9 14, 18, 1D) _{, and} 10? for bean

in -R.solanj infested sand (f ig l-C) .

Trichoderma treatment oi unprimed seeds did not rnodify

plant stands for lettuce (in Pythium-infested sand), or

increased it slightly for all other crop

pathogen-combinations, compared to untreated seeds'

i" eytiiun-infested sand, emergence of _-unprirned seeds

compared io primed seeds ranged from 262 to 66e. for cucutr.ber,

frorn fe2 to 1AZ for bean, and frorn 262 Lo 622 for lettuce. In

R. so-l.anj inf ested sand, prirning also increased bean seeds emergence _'Connininq (from 10? with unprimed seeds to 88%) '

seed prining with Trichoderna treatment resul-ted

in an increased _"^"ig.r,"" for cucumber-pythiurn or bean-Pythiun combinations _{' For} éucumber, stands rànged fron 262 with

untreated seeds to 882 with Trichoderma treatment of primed

seeds. For bean, stands ranged from 162 with untreated seeds

l" ôAZ with ?rjchoderma trealment of primed seeds. In

lettuce-pvthium and bean-Rà-izoctonia combinations, Trichoderma

lià.i*."t did not provide any additional emergenee, but iËàùààa damping_off postemergence symptoms.,co_nversely, in uninfested =and, unprimed, Tiichoderna-treated fettuce seeds resulted in more râpid emerqence than untreated seeds' and pi""t height and general robutness were improved (Fj-g' 2)

Trichoderma population densitv on seeds

PopuJ-ations of Trichodetna in cucumber or bean seeds was

evaluated before and after drying prined Tricboderna-treated seeds. In cucumber seeds, poputatign density onf Trichoderma decreased after drying, from 47 x l-Oq to 17 x 10- CFU per gram àtV-t.ignt 1aa? reduc-tion;. Population of Trichoderma _-in bean

="âd= dÉclined after dryinq frbm an inj"tial leveI of 1o xLo'

t;-il * ro3 cFU per gram dry weight (24% reduction) _' - --uÀwever, _{ki1lin; Tric-hoderna by Sumico}

-treatment after priming, did not modi-fy the protection obtained (Fig' 3)

DISCgSSION

Matrix prining markedly protected seedlings.of alI crops

.==uyàà àgain=t eytniun ..ta/-ot R'so-lani ' Benef its resulting

f rom the ,.,=" of matrix priming, could be explained by the

iàf".=" of seed exudates'during prining. Osburn et a7' (15,

iài- _"À".""a that the rate of exudation by sugar beet seeds

â"i';-ts washing and osmopriming, was correl-ated w j-th the rate

of germination in soit infested with Pythlum-. Elirni-nation of qàr;ination j_nhibitors present in seed coats by washing seems

ât=o to be related to damping-off control (14) ' other explanation coufd be that, due to increased rate of energence

u"à growth, seedli_ng rnight escape to lethal infection by

Pythiun or _' Rhizoctonia.

?h. protection of seedlings by Trichoderma appears to be dependent on the delivery system' In infested sand,

Trichoderna seed coating of unprj.rned seeds increased emergence

sllghtlyforbean,significantlyforcucumber,butdidnot

p.o.iid"' any protection for lettuce. Trichoderna enhanced the

positive

"if""t of prirning in terms of emergence in cucunber-'eythiun and bean-tythium -seeds,. While improving plant vigour

rn eytniun-lettuce -and Rhizoctonja-bean combinations'

There was a marked increase j-n the growth rate of Trichoderna-treated Iettuce seeds in uni-nfested sand. It was

vj-sually apparent, as the plants emerged rapi-d1y and continued

t; ;;;; thiough out the pàriod of the experiment' Àhmad and gakJr (1), found increased growth responses of plants as a consequànce of seed treatment with Trichoderma on cucumber,

pea, iomato and radish. Windhrnan et al' (20) found that the

iate of tornato and tobacco gernination was i-ncreased, conpared

to controls where TrlchoderÀa spp, \,ias separated from seeds by

aceIJ-ophanemembrane.TheyconcludedthatsuchTrichoderma spp. pràduced a growth-regulating factor thât increased the rate of seed germination.

our data on population dynamics of Trichoderna before and after ar'i"q primàa^seeds, sh-owed that the fungus populatj-ons declined after drying, while killinq Trichoderma, by a

fungj-cide treatment-appfl""a after. prininq-, did not modify the

oroÉection observed. -T-h,-,=, col-onization by, and activity . of _'

'r;l;i;à;;;a _{during the precolonization period' i-s sufficient}

i;----;6;;== its proÈective abiritv, Iinked to site

occupation ( a ) , renova-l of exudates (6, L'l , .:-8) , production of toxià metatot'ites (12, 13) and/or hydrolytic enzymes (3, 5)' rtre ract that kil1i.ng Trichaderma after prinj-ng did not affect

lirÀ pi"tecti.re etteJt, excludes mycoparasitism as a mode of action.

oneofthelirrritingfactorsinthedevelopment.of

biological control producÉs has been thej-r 1ow reliabiJ-ity, variaÉle efficacy anà narrow spectrum of activity'

prirning seeàs in the presence of Triehoderma spores seems

an attracti-ve method of protection against damping:off agents;

it promoted reproducible effects in terrns of high emergience p".Ë"r.,tuq., imp^rove the vigour of the plants and reduced

post-à*".g..,"é s1 ptoms, with -several crop-pathogen _{.combinations.}

;;;;'; praciiàar poj-nt of vj-ew, priming avoids- the problem of

survivai of the biocontrol agent in treated seeds' LITERÀTURE CIÎED

1- Àhmad, J. J. _{, and Baker, R'} 1988 _{' .Irnplications} of

rhizosphere competencé of Trichoderna harzianun' Canadian Journal- of Microbiology 34| 229-234,

2- Bradf ord, X.- ,l . 1984. Seed pr j-ning: techniques to speed seed germination. Proc.oregon Hortic.soc 25r 227-233.

3- ch;t, I., Harnanf G. E', and Baker, R' 1981-' Trichoderma hanatun: It/s hyphal interactions with Rhizoctonia

so-lani and Pythrum spp. Micrcbiol. Ecol' 7: 29-38'

4- Cotes, A. M., Lepoivre, P', and Semal--, J' i992'

Effect of precolonization of bean seeds lvith Trichodetna' on =y*pt"m=induceabyPythiun.Med.Fac.Landbowv.Univ'Gent_{5t /2b.. 355-363.}

t326

1m _{Cucumber, Pythium}

Bean, Pythium

Bean, R. solani

6

Days after planting

Fig 1. Emergence percentage of (A) cucumber, (B,C) or bean gràwn from unpri.mede--o, primeda-4, unprined+Trichoderlûâ*-'-* t

ànd prinred+T;ichoderma'e-.€ seeds. seeds were planted in sand

artiiicially infested wj-th Pythiun, (cucumber, bean and

J-ettuce) or with R. solanj (bean seeds). The bars represent the

standard deviation about each point.

8æ o Iso @ Eao U f. eo o c^^ o o Eao I! à9 so o o o E{ U

€

O

o

Fig 2. Emergence uninfested sand,

Trichodermâe

DaYS atter Planting

percentage of unPrirned

untreated control- F Iettuce seeds inor treated with

a;'1oo E,o_c o ?æ o E u40 æ 12

æ

Fig 3. Effect of kiJ-ling Trichoderma after prirning on the

pr6tection against Pythiun (A) cucumber seeds (B) bean seeds

Seeds were treatea witn vrater _S (Trichoderma stil1 ative) or

5- Elad, Y., Chet, I.,and Henis, Y. l-982. Degradation of plant pathogenic fungi by Trichoderna harzianum. Canadian

Journal of Microbiology 28r 7L9-725.

6- Hadar, I., Chet, L, and Hennis, Y. 1979. Biological

control of Rhizoctonia solani dampj-ng-off with wheat bran

culture of Trichoderma harzianum. Phytopathology 69. 64-68.

7- Harman, G. E, _{, and Taylor, A. G. 1988.} Improved

seedling performance by integration of biologicaJ- control agents at favorable pH IeveIs with solid matrix prirni-ng.

Phytopathology 78t 520-525.

8- Harman, G. 8., Taylor, A. G., and Stasz, T. E. 1989.

Conrbining effective strains of Trichoderma harzianutn and solid matrix priming to irnprove biological seed treatments. Plant

Disease 73263I-637.

9- Heydecker, W. _, Hj"ggins, J. _, and Gul-l-iver, R. L. L973.

Àccelerated gerrnination by osmotic seed treatment. Nature 2461 42-44 .

1O- Heydecker, W. L97 4 . Gerrnlnatj-on of an idea: the prinring of seed. Univ.Nottinqharn School Àgric.Rep. 1973/79741 50-67 .

11- Heydecker, W, Higgins, J., and Turner, Y. J. a975,

Invigoration of seeds? Seed Sci.Tech. 3: 881--888.

L2- Lewj-s, J. A. _{, and Papavizas, G. c. 1984. À} nev/ approach to stirnulate population proliferation of Trichoderna

species and other potentj.al biocontrol fungj- introduced into natural soils. Phytopathology 74| L240-l.244.

l-3- Lif shitz, R. _, Windham, M. T. _{, and Baker, R.} 1986.

Mechanism of biologicaL control of preemergence damping-off of pea by seed treeatment wj-th Trichoderma sPp. Phytopathology

'76i _720-725.

14- Murray, G, À., Gallian, J' J. _{, Kay, M.} À. _, and Stewart, K, L987. Seedling emergence from preconditioned sugar

beet seed. Univ. Idaho Winter Conrnodity School-s 1-9r 21-3-21-6. 15- osburn, R. M., and Schroth, M. N. 1988. Effect of osnopriming sugar beet seed on exudation and subsequent

darnping-off caused by Pythiun u-ltinum. Phytopathology 78. 1246-1250.

16- osburn, R. M., and Schroth, M. N. 1989. Effect of

csmoprining sugar beet seed on germination rate and incidence

of Pythiun ul.timum danping-off. 19E9. Plant Disease 73. 2L-24.

l-7- Schroth, M. N., and cook, R. J. 7964. Seed exudation

and its influence on pre-emergence danping-off of bean. Phytopathology 54 | 6'7 0-6'7 3 .

18- Short, G. 8., Loria, R,, and Lacy, M. L.

Factors affecting pea seeds and seedling rot in

Phytopathology 66: l-88-l-92 ,

1977 .

soi1.

19- Weinhold, À. R., Bowman, T,, and Dodman, R. L. 1969.

Virulence of Rhizoctonia so-lanj as affected by nutritj-on of the pathogen. Phytopathology 59: 1601-1605,

20- Windham, M. T., E1ad, Y., and Bakerr R. 1986. A mechanism for increased plant growth induced by Triclloderma spp. Phytopathology 76. 57A-52!.