Suppression of Botrytis cinerea causing the grey mould disease ofgrape-vine by an aggressive mycoparasite, Pythium radiosum

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

Received 2 April 1999; received in revised form 23 April 1999; accepted 26 April 1999

Abstract

Pythium radiosum Paul has been found to be an aggressive mycoparasite of Botrytis cinerea which causes grey mould diseaseon the grape-vine. The mycoparasitic fungus enters the host mycelium, coagulates its protoplasm, empties its contents andfinally comes out producing numerous ramifications and sexual structures. When the infected mycelium of B. cinerea is appliedto the leaves of the grape-vine, the characteristic grey mould symptoms fail to appear. Since P. radiosum causes no harm to thegrape-vine, it can be used as a biological control agent against B. cinerea. A brief account of the mycoparasite and itsantagonism towards B. cinerea is discussed here. z 1999 Federation of European Microbiological Societies. Published byElsevier Science B.V. All rights reserved.

Keywords: Botrytis cinerea ; Mycoparasite ; Biological control ; Pythium radiosum ; Antagonism

1. Introduction

Mycoparasites are fungi that can parasitize otherfungi. This term is generally used to include parasitesthat coil around the host hyphae or overgrow othercolonies on agar. This may involve either penetrationof the host hyphae or antagonism by the productionof antibiotics [4], toxic radicals [10], or wall-lytic en-zymes [2]. The phenomenon of mycoparasitism isvery widespread in nature. A review of the literatureon this important subject has already been published[1,8,11,17,18]. Most evidence for the role of myco-parasitism comes from the observation of infectedfungal propagules such as spores or other reproduc-

tive structures. Oospores of Aphanomyces, Phytoph-thora, Pythium, and Sclerospora have been found tobe infected by a wide range of fungi including Fusa-rium mesmoides, Hyphochytrium catenoides, Olpidiop-sis gracilis, Dactyella spp. and Trinacrium subtile[16]. Facultative mycoparasites such as Trichodermaand Gliocladium species have been reported from thesclerotia of di¡erent parasites like Phymatotrichumomnivorum, Rhizoctonia spp., Sclerotinia spp., andVerticillium dahliae [17]. Unfortunately this phenom-enon of mycoparasitism has not been much ex-ploited. Some of the mycoparasites that have beenused as biological control agents are Ampelomycesquisqualis for the control of cucumber powdery mil-dew caused by Sphaerotheca fuliginea [7] and Erysi-phe cichoracearum [15] under glasshouse conditions.Mycoparasitism of Trichoderma harzianum has been

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studied extensively. This mycoparasite is antagonisticand e¡ective in controlling plant pathogenic fungilike Rhizoctonia solani, Sclerotium rolfsii, Fusariumoxysporum, Fusarium culmorum, and Pythium apha-nidermatum [3]. It has also been widely studiedagainst Botrytis cinerea causing the grey mould dis-ease of grape-vine and Chondrostereum purpureum,the agent of silver leaf in fruit trees [5].

Pythiaceous fungi are also known to be mycopar-asites of a wide range of fungi. The most studiedmycoparasite of the genus or perhaps of all myco-parasites known is Pythium oligandrum Drechsler. Ithas a very wide host range and rapidly attacks thesusceptible hosts by lysis or penetration of hyphae[12]. However, it is only one of six known mycopar-asitic Pythium species, the others being Pythiumacanthicum Drechs., P. acanthophoron Sideris, P. my-coparasiticum Deacon et al., P. periplocum Drechs.,and P. nunn Lifshitz et al. [9]. Apart from these wellknown mycoparasites, one aggressive plant patho-gen, P. aphanidermatum (Edson) Fitzpatrick, hasalso been studied for its mycoparasitic activity [9].

Pythium radiosum was isolated in 1992 from a soilsample collected in the north of France and de-scribed as a new species by the author [14]. Sincethen it has not been reported from anywhere elsein the world. It has echinulate oogonia resemblingPythium echinulatum Matthews. When grown togeth-er with B. cinerea, it rapidly enters the latter's myce-lium and causes extensive damage. This is the ¢rstreport of the mycoparasitism of P. radiosum.

2. Materials and methods

P. radiosum (F-12) was re-cultured from the au-thor's personal collection of pythiaceous fungi, main-tained at the `Institut Jules Guyot', in Dijon, France.B. cinerea (B-3) was also obtained from the culturecollection of this institute. Both fungi were main-tained on PDA (potato dextrose agar), and PCA(potato carrot agar) media. P. radiosum was alsomaintained on boiled hemp-seed halves in sterile dis-tilled water. Inter-hyphal interactions were studiedby placing both fungi on the same PDA plate onopposite ends (Fig. 2a) and also on a thin ¢lm ofPDA on a glass slide (Fig. 2b) which was obtained asfollows.

A glass rod was heated and bent in a `V' shape insuch a way that it could enter a glass petri dish of 10cm diameter. It was introduced into the petri dishand a glass slide was placed on the top of the Vrod. The whole was autoclaved. A thin layer ofPDA was poured aseptically on the glass slide andthe two fungi were inoculated at the opposite ends ofthe PDA ¢lm. To maintain humidity, enough steriledistilled water was poured in the petri dish so thatthe water remained well below the slide. Three rep-licates were prepared in the same manner and allwere incubated at 25³C for 15 days. From time totime sterile distilled water was added to the petridishes in order to avoid dehydration of the thinPDA ¢lm. After 15 days a small square of thePDA ¢lm was aseptically cut and placed on a freshslide in a drop of sterile water. A coverslip wasplaced on the ¢lm and this was sealed with nail pol-ish to avoid dehydration. The slide was then ob-served by microscope.

Inoculation experiments with B. cinerea were car-ried out on the leaves of 1-month-old Vitis viniferavitro cultures maintained in our laboratory. Theleaves were taken out aseptically and were intro-duced into three sterile petri dishes having a sterile¢lter paper. Sterile distilled water was used to humid-ify the ¢lter paper. One leaf was placed in each petridish. The leaf of the ¢rst petri dish was inoculatedwith 50 Wl sterile distilled water in which B. cinereamycelium from a 1-week-old PDA culture was intro-duced. The leaf of the second petri dish was inocu-lated with 50 Wl of sterile distilled water in which theparasitized mycelium (with P. radiosum) was placed,and the leaf in the third petri dish was inoculatedwith 50 Wl of sterile distilled water in which the my-celium of P. radiosum was introduced. All three petridishes were maintained at room temperature (20³C).Occasionally the ¢lter papers of the three dishes werehumidi¢ed with sterile distilled water. This experi-ment was repeated thrice.

3. Results

3.1. Morphological description

P. radiosum (Fig. 1a^e): mycelia are well branchedat times bearing conical spines measuring 3^7 Wm in

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Fig. 1. Morphological structures of P. radiosum. a^c: Oogonia ornamented with conical to mammiform spines. d: Smooth-walled sporan-gia. e: Scanning electron micrograph of an ornamented oogonium. Bar: a^d = 25 Wm, e = 10 Wm.

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Fig. 3. Hyphal interactions between B. cinerea and P. radiosum. a: Normal hypha of B. cinerea. b: Hypha of P. radiosum (mycoparasite)inside the mycelium of B. cinerea (host). c: Coagulated protoplasm in the hypha of B. cinerea. d: Proliferation and exit of P. radiosumhyphae from the hypha of B. cinerea. e : Emptied hypha of B. cinerea. f : Damaged hypha of B. cinerea with sexual structure of P. radio-sum. Bar: a^c, e, f = 35 Wm, d=100 Wm.

Fig. 2. Interaction between B. cinerea (B-3) and P. radiosum (F-12). a: Antagonism between B-3 and F-12 on PDA plate. b: Antagonismbetween B-3 and F-12 on a ¢lm of PDA on a slide.

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length. Sporangia are mostly intercalary measuring6^30 Wm in diameter and are mostly non-sporulating(Fig. 1d). Oogonia are mostly intercalary and cate-nulate and are covered with conical to mammiformspines (Fig. 1a,b,c,e). Oogonia (excluding spines) are10^35 Wm in diameter and are provided with spineswhich can measure up to 15 Wm long and up to 6 Wmthick at the base. Antheridia are rare and whenpresent are hypogynous. After fertilization the oo-spores produced are aplerotic with a relatively thinwall.

In the hyphal interaction experiments, the coloniesof P. radiosum (F-12) which are whitish in colorspread into the greyish colonies of B. cinerea (B-3).The latter does not spread into the former's zone(Fig. 2a,b). Microscopic observation of the myceliumin the zone of contact reveals that P. radiosum entersfreely into the mycelium of B. cinerea (Fig. 3b^d)and once inside it rami¢es (Fig. 3b), the rami¢ca-tions enter the septa of the fungal hyphae thus pass-ing from one cell to another without any hindrance.At ¢rst the cytoplasm of the host cell is coagulated(Fig. 3c) and then the mycoparasite consumes thecytoplasm leaving an empty host mycelium (Fig.3e) and ¢nally the mycoparasite comes out forminghere and there an entangled mass of its myceliumaround the host (Fig. 3d) and reproductive structures(Fig. 3f).

After inoculation on the leaf the vitro plants of V.vinifera showed typical grey mould symptoms on theleaf of the ¢rst petri dish (Fig. 4a), no disease on theleaf of the second petri dish (Fig. 4b) and also noinfection on the third. All three replicates showed thesame results.

4. Discussion

The experiments show that the mycelium of P.radiosum can rapidly enter the hyphae of B. cinerea.Upon entry, the fungus rami¢es within and provokeslarge-scale destruction of the host mycelium. Hencethe former is undoubtedly a mycoparasite of the lat-ter. Since the parasitized mycelium of B. cinerea failsto produce the characteristic grey mould symptomson the leaves of V. vinifera, and since the mycopar-asite does not harm the leaves of the grape-vine inany way, P. radiosum can be used as a biocontrolagent against B. cinerea.

The mode of action of P. radiosum di¡ers fromthat of P. oligandrum which often causes explosivelysis of host mycelium [12], whereas P. radiosumbrings about cytoplasmic coagulation like that ob-served in the case of P. aphanidermatum [9]. Thefact that P. radiosum penetrated the host myceliumand grew extensively within it may be useful in the

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Fig. 4. Inoculation experiments on V. vinifera. a : Symptoms of grey mould disease on V. vinifera caused by B. cinerea. b: Healthy leaf ofV. vinifera inoculated with parasitized B. cinerea with P. radiosum.

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analysis of the mechanisms of mycoparasitism, espe-cially the suggested roles of cell wall-degrading en-zymes [2,6,13].

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