Pythium periplocum, an aggressive mycoparasite of Botrytis cinereacausing the gray mould disease of grape-vine

Bernard Paul *Laboratoire des Sciences de la Vigne, Institut Jules Guyot, Universite de Bourgogne, B.P. 138, 21004 Dijon, France

Received 12 October 1999; received in revised form 17 October 1999; accepted 19 October 1999

Abstract

Pythium periplocum Dreschler has been found to be an aggressive mycoparasite of Botrytis cinerea, the causal agent of thegray mould disease of the grape-vine. When grown together, the former enters the latter's mycelium, branches freely within,coagulates its cytoplasm and finally tears its hyphae apart, bringing about widespread destruction of the grape-vine pathogen.Extensive coiling around the host, as reported in the case of other mycoparasites belonging to the genus Pythium, has not beenobserved here. The infected mycelium of B. cinerea fails to infect the grape-vine and does not induce the characteristic graymould symptoms. Since P. periplocum is not a grape-vine parasite, it could be useful for the biological control of B. cinerea. Abrief account of this mycoparasitism is discussed in this article. ß 1999 Federation of European Microbiological Societies.Published by Elsevier Science B.V. All rights reserved.

Keywords: Mycoparasite; Fungus; Mycelium; Biofungicide; Pythium periplocum ; Botrytis cinerea

1. Introduction

Mycoparasitism is a very common phenomenonexisting naturally in nature. Quite a lot of interestingliterature already exists regarding this subject [1^5].A mycoparasite is a fungus which attacks other fun-gi. When grown together, it can overgrow the otherfungus, its mycelium may coil around the host my-celium, it can also show antagonism to the host fun-gus by secreting antibiotics [6], toxic radicals [7] orwall lytic enzymes [8]. It can also enter the host my-celium, bringing about coagulation of the host pro-toplasm and ¢nally causing widespread damage to

the latter [9]. Mycoparasitism by the members ofthe genus Pythium is also well known, the most im-portant fungi being Pythium oligandrum and Pythiumacanthicum [10^13]. Pythium periplocum has alsobeen considered as a mycoparasite and recently, to-gether with P. oligandrum and P. acanthicum, it wasstudied on 73 species of ¢lamentous fungi and acomparison of the mycoparasitism of the three spe-cies was made [14]. Apart from these three species ofPythium, there are sporadic reports of mycoparasi-tism by other species like Pythium acanthophoron [15]and Pythium radiosum [9]. All these fungi have onecommon structural resemblance: their oogonial wallsare ornamented with spines. Very rarely, a fungus ofthe genus Pythium having smooth walled oogoniahas been reported to be a mycoparasite. One suchexample is that of Pythium aphanidermatum, which

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* Corresponding author. Tel. : +33 (3) 80396341;Fax: +33 (3) 80396326; E-mail: bernard.paul@u-bourgogne.fr

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has been reported to antagonize P. oligandrum bycoiling around and penetrating into the latter's hy-phae [10]. Out of all these fungi, only P. oligandrumand P. acanthicum are established mycoparasites [10^13,16,17]. P. periplocum is a relatively less well-known fungus in this ¢eld, mainly because it is notvery widely distributed. It was originally isolatedfrom Citrullus vulgaris in the USA [18] and eversince, it has been reported from a limited numberof geographic locations (Argentina, Brazil, Sudanand The Netherlands) [19]. It has been used as abiological control agent for the damping-o¡ of cu-cumber seedlings caused by P. aphanidermatum [20].A recent report indicates that it can parasitize anumber of host fungi, coiling around their hyphaeor even penetrating within. It has been considered tobe a broad-spectrum mycoparasite at par withP. oligandrum and P. acanthicum [14].

In this study, it was found that P. periplocum wasan aggressive mycoparasite of Botrytis cinerea, whichcauses the gray mould disease of the grape-vine.However, the typical coiling around the host hyphaewas never found. On the contrary, penetration byP. periplocum into the Botrytis mycelium and wide-spread destruction were frequently observed.

Fig. 1. P. periplocum. a : ¢lamentous in£ated sporangia, b: orna-mented oogonia with antheridia, c: almost plerotic oospore, d:aplerotic oospore. Bar (a) = 40 Wm, (b^d) bar = 20 Wm.

Fig. 2. B. cinerea. a: Conidia and healthy hyphae, b: hyphae infected with P. periplocum, c,d: coagulated cytoplasm of hyphae infectedby P. periplocum. Bar (a,b,d) = 20 Wm, (c) bar = 40 Wm.

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2. Materials and methods

P. periplocum (CI-056) and B. cinerea (BC-3) weretaken from the author's personal collection of ¢la-mentous fungi maintained at the `Institut JulesGuyot', Dijon, France. Both the fungi were main-tained on potato dextrose agar (PDA). Antagonismbetween the two was observed by placing them onthe same PDA plate at opposite ends and also on athin PDA ¢lm on a glass slide as described earlier [9].Three replicates were prepared for all the plates andslides and were incubated at 25³C for a period of2 weeks. Sterile distilled water was poured occasion-ally on the PDA ¢lm on the slide to avoid dehydra-tion. After the incubation period, a small piece of thePDA was aseptically cut from the PDA plate and¢lm and placed on a slide in a drop of sterile distilledwater and sealed under a cover-slip for microscopicexamination. Inoculation experiments with healthyand mycoparasitized B. cinerea mycelium were per-formed on 2-months-old grape-vine plants (Vitis vin-ifera), cultivars `Chardonnay' and `Pinot noir', asdescribed earlier [21].

3. Results

P. periplocum was identi¢ed by the keys providedby Plaatts-Niterink [19] and Dick [22]. It is an easilydistinguishable fungus because it is the only memberof the genus Pythium showing the characteristic com-bination of having ornamented oogonia (Fig. 1b^d)and a ¢lamentous in£ated type of sporangia (Fig.1a). In the interaction experiments between P. peri-plocum and B. cinerea, the extensive hyphal coilingsaround the host hyphae as reported elsewhere [14]have not been observed in the present study. How-ever, direct penetration into the host hyphae by themycoparasite (Figs. 2b and 3a) and coagulation ofthe Botrytis protoplasm have been observed. P. peri-plocum enters easily the mycelium of B. cinerea, itrami¢es therein (Figs. 2b and 3b), coagulates thehost protoplasm (Fig. 2c,d) and ultimately emptiesthe latter's contents. Finally, the mycoparasiteemerges out by tearing apart and completely destroy-ing the host mycelium (Fig. 3c,d). The infected my-celium of B. cinerea failed to bring about the graymould symptoms on either of the two cultivars(Chardonnay and Pinot noir) of grape-vine vitroplants.

Fig. 3. Interaction between B. cinerea and P. periplocum. a,b: Entry and rami¢cation of mycoparasite (P. periplocum) into the host (B.cinerea) mycelium, c,d: destruction of host mycelium and emergence of the mycoparasite. Bar (a) = 40 Wm, (b^d) bar = 20 Wm.

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4. Discussion

P. periplocum is not a very common fungus. It hasornamented oogonia and a ¢lamentous in£ated typeof sporangia. It was ¢rst described as a pathogen ofC. vulgaris L. in the USA causing the disease ofblossom-end rot [18]. Subsequently, it has been de-scribed sporadically from diseased plants or from thesoil. P. periplocum has not been extensively reportedto be a mycoparasite like P. oligandrum and P. acan-thicum. However, a few studies reveal the mycopar-asitic ability of this fungus [12,20]. A recent reportsuggests that P. periplocum is an aggressive mycopar-asite with a wide host range comparable with con-generic broad-spectrum mycoparasites like P. oligan-drum and P. acanthicum [14]. Our study shows thatP. periplocum is an aggressive mycoparasite ofB. cinerea and its aggressiveness to this fungus iscomparable to that of P. oligandrum and P. acanthi-cum (unpublished). Extensive coiling around the hosthyphae as reported elsewhere [14] has not been ob-served in our study of its attack on B. cinerea. Onthe other hand, entry of the mycoparasite into thehost hyphae, coagulation of the host protoplasmand, ultimately, killing and tearing o¡ of the latterhas been frequently observed. The infected myceliumof B. cinerea is devoid of its pathogenic capabilitieson V. vinifera and since P. periplocum is not harmfulto the grape-vine plant, it can also be used as a bio-logical control agent like P. oligandrum. However,¢eld trials and formulations have to be studied priorto its practical use as a biofungicide.

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