Saprophytic microorganisms with potential for biological control of Botrytis cinerea on Geraldton waxflower flowers

Saprophytic bacteria, yeasts and filamentous fungi were isolated from Geraldton waxflower flowers and screened to identify potential antagonism towards Botrytis cinerea. Isolates from other sources (e.g. avocado) were also tested. Isolates were initially screened in vitro for inhibition of B. cinerea conidial germination, germ tube elongation and mycelial growth. The most antagonistic bacteria, yeasts and fungi were selected for further testing on detached waxflower flowers. Conidia of the pathogen were mixed with conidia or cells of the selected antagonists, co-inoculated onto waxflower flowers, and the flowers were sealed in glass jars and incubated at 20 degreesC. The number of days required for the pathogen to cause flower abscission was determined. The most antagonistic bacterial isolate, Pseudomonas sp. 677, significantly reduced conidial germination and retarded germ tube elongation of B. cinerea. None of the yeast or fungal isolates tested was found to significantly reduce conidial germination or retard germ tube elongation, but several significantly inhibited growth of B. cinerea. Fusarium sp., Epicoccum sp. and Trichoderma spp. were the most antagonistic of these isolates. Of the isolates tested on waxflower, Pseudomonas sp. 677 was highly antagonistic towards B. cinerea and delayed waxflower abscission by about 3 days. Trichoderma harzianum also significantly delayed flower abscission. However, as with most of the fungal antagonists used, inoculation of waxflower flowers with this isolate resulted in unsightly mycelial growth.

CHARACTERISATION OF ARABIDOPSIS THALIANA PERMEABLE CUTICLE MUTANTS SHOWING A STRONG RESISTANCE TO BOTRYTIS CINEREA

La cuticule des plantes, composée de cutine, un polyester lipidique complexe et de cires cuticulaires, couvre l'épiderme de la plupart des parties aériennes des plantes. Elle est constituée d'une barrière hydrophobique primaire qui minimise les pertes en eau et en soluté et protège l'organisme de différents stress environnementaux tels que les rayons UV, la dessiccation et l'infection par des pathogènes. Elle est aussi impliquée dans la délimitation des organes durant le développement. La cutine est un polyester qui, dans la plupart des espèces végétales, est principalement composé d'acides gras ω-hydroxylés composé de 16 à 18 carbones. Cependant, la cutine des feuilles d'Arabidopsis a une composition différente et est principalement constituée d'acides dicarboxyliques à 16-18 carbones. Les cires sont présentes dans le polyester de la cutine ou le recouvrent. Chez Arabidopsis, un nombre de mutants, tel que 1er, bdg, hth, att1, wbc11, et des plantes transgéniques avec différents changement dans la structure de la cuticule dans les feuilles et la tige, ont récemment été décrits et servent d'outils pour étudier la relation entre la structure et la fonction de la cuticule.7 mutants d'Arabidopsis ont été isolés par une méthode de coloration qui permet de détecter une augmentation dans la perméabilité cuticulaire. Ces mutants ont été appelés pec pour permeable cuticle.Pour la première partie de mon projet, j'ai principalement travaillé avec pec9/bre1 (permeable cuticle 9/botrytis resistance 1). PEC9/BRE1 a été identifié comme étant LACS2 (LONG CHAIN ACYL-CoA SYNTHETASE 2). Dans ce mutant, la cuticule n'est pas visible sous microscopie électronique et la quantité en acides gras omega- hydroxylés et en leurs dérivés est fortement réduite. Ces altérations conduisent à une plus grande perméabilité de la cuticule qui est mise en évidence par une plus grande sensibilité à la sécheresse et aux xénobiotiques et une coloration plus rapide par bleu de toluidine. Le mutant Iacs2 démontre aussi une grande capacité de résistance à l'infection du champignon nécrotrophique B. cinerea. Cette résistance est due à l'extrusion sur les feuilles d'un composé antifongique durant l'infection. Ce travail a été publié dans EMBO journal (Bessire et al., 2007, EMBO Journal).Mon second projet était principalement concentré sur pec1, un autre mutant isolé par le premier crible. La caractérisation de pec1 a révélé des phénotypes similaires à ceux de Iacs2, mais à chaque fois dans des proportions moindres : sensibilité accrue à la sécheresse et aux herbicides, plus grande perméabilité au bleu de toluidine et au calcofluor white, altération de la structure cuticulaire et résistance à B. cinerea à travers la même activité antifongique. PEC1 a été identifié comme étant AtPDR4. Ce gène code pour un transporteur ABC de la famille PDR ("Pleiotropic Drugs Resistance") qui sont des transporteurs ayants un large spectre de substrats. Le mutant se différencie de Iacs2, en cela que la composition en acides gras de la cuticule n'est pas autant altérée. C'est principalement le dihydroxypalmitate des fleurs dont la quantité est réduite. L'expression du gène marqué avec une GFP sous le contrôle du promoteur endogène a permis de localiser le transporteur au niveau de la membrane plasmique des cellules de l'épiderme, de manière polaire. En effet, la protéine est principalement dirigée vers l'extérieure de la plante, là où se trouve la cuticule, suggérant une implication d'AtPDR4 dans le transport de composants de la cuticule. Ce travail est actuellement soumis à Plant Cell.Une étude phylogénétique a aussi montré qu'AtPDR4 était très proche d'OsPDR6 du riz. Le mutant du riz a d'ailleurs montré des phénotypes de nanisme et de perméabilité similaire au mutant chez Arabidopsis.AbstractThe cuticle, consisting principally of cutin and cuticular waxes, is a hydrophobic layer of lipidic nature, which covers all aerial parts of plants and protects them from different abiotic and biotic stresses. Recently, the research in this area has given us a better understanding of the structure and the formation of the cuticle. The Arabidopsis mutants permeable cuticle 1 (peel) and botrytis resistance 1 (brel) were identified in two screens to identify permeable cuticles. The screens used the fluorescent dye calcofluor to measure permeability and also resistance to the fungal pathogen Botrytis. These mutants have highly permeable cuticle characteristics such as higher water loss, intake of chemicals through the cuticle, higher resistance to Botrytis cinerea infection, and organ fusion.BRE1 was cloned and found to be LACS2, a gene previously identified which is important in the formation and biosynthetic pathway of the cuticle. In brel, the amount of the major component of cutin in Arabidopsis leaves and stems, dicarboxylic acids, is five times lower than in the wild type. Moreover, the permeability of the cuticle allows the release of antifungal compounds at the leaf surface that inhibits the growth of two necrotrophic fungi: Botrytis cinerea and Sclerotinia sclerotiorum.PEC1 was identified as AtPDR4, a gene that codes for a plasma membrane transporter of the Pleiotropic Drug Resistance family, a sub-family of the ABC- transporters. AtPDR4 is strongly expressed in the epidermis of expanding tissues. In the epidermis it is located in a polar manner on the external plasma membrane, facing the cuticle. Analysis of the monomer composition of the cutin reveals that in this mutant the amount of hydroxy-acids and dihydroxy-palmitate is 2-3 times lower in flowers, in which organ these cutin monomers are the major components. Thus AtPDR4 is thought to function as a putative cutin monomer transporter.

A permeable cuticle in Arabidopsis leads to a strong resistance to Botrytis cinerea.

The plant cuticle composed of cutin, a lipid-derived polyester, and cuticular waxes covers the aerial portions of plants and constitutes a hydrophobic extracellular matrix layer that protects plants against environmental stresses. The botrytis-resistant 1 (bre1) mutant of Arabidopsis reveals that a permeable cuticle does not facilitate the entry of fungal pathogens in general, but surprisingly causes an arrest of invasion by Botrytis. BRE1 was identified to be long-chain acyl-CoA synthetase2 (LACS2) that has previously been shown to be involved in cuticle development and was here found to be essential for cutin biosynthesis. bre1/lacs2 has a five-fold reduction in dicarboxylic acids, the typical monomers of Arabidopsis cutin. Comparison of bre1/lacs2 with the mutants lacerata and hothead revealed that an increased permeability of the cuticle facilitates perception of putative elicitors in potato dextrose broth, leading to the presence of antifungal compound(s) at the surface of Arabidopsis plants that confer resistance to Botrytis and Sclerotinia. Arabidopsis plants with a permeable cuticle have thus an altered perception of their environment and change their physiology accordingly.

Enhanced Botrytis cinerea resistance of Arabidopsis plants grown in compost may be explained by increased expression of defense-related genes, as revealed by microarray analysis

Composts are the products obtained after the aerobic degradation of different types of organic matter waste and can be used as substrates or substrate/soil amendments for plant cultivation. There is a small but increasing number of reports that suggest that foliar diseases may be reduced when using compost, rather than standard substrates, as growing medium. The purpose of this study was to examine the gene expression alteration produced by the compost to gain knowledge of the mechanisms involved in compost-induced systemic resistance. A compost from olive marc and olive tree leaves was able to induce resistance against Botrytis cinerea in Arabidopsis, unlike the standard substrate, perlite. Microarray analyses revealed that 178 genes were differently expressed, with a fold change cut-off of 1, of which 155 were up-regulated and 23 were down-regulated in compost-grown, as against perlite-grown plants. A functional enrichment study of up-regulated genes revealed that 38 Gene Ontology terms were significantly enriched. Response to stress, biotic stimulus, other organism, bacterium, fungus, chemical and abiotic stimulus, SA and ABA stimulus, oxidative stress, water, temperature and cold were significantly enriched, as were immune and defense responses, systemic acquired resistance, secondary metabolic process and oxireductase activity. Interestingly, PR1 expression, which was equally enhanced by growing the plants in compost and by B. cinerea inoculation, was further boosted in compost-grown pathogen-inoculated plants. Compost triggered a plant response that shares similarities with both systemic acquired resistance and ABA-dependent/independent abiotic stress responses.

Potencial para o biocontrole de Botrytis cinerea por leveduras em sistema integrado de cultivo de lírio

O objetivo deste trabalho foi comparar uma população de leveduras do filoplano de lírio de um sistema convencional de cultivo com outra de sistema integrado, além de avaliar o antagonismo da levedura Sporidiobolus pararoseus a Botrytis cinerea. A comunidade de leveduras foi estimada pelo método da queda dos balistoporos nos terços inferior, médio e superior das plantas, bem como nas faces adaxial e abaxial das folhas. Nos discos de folhas de plantas do sistema integrado, foi alta a presença da população de leveduras, tendo sido maior na face adaxial das folhas do terço médio das plantas. Para avaliação do antagonismo, foram aplicadas aos discos de folhas as concentrações de 10(5), 10(6) e 10(7) células mL-1 da levedura, em três períodos de inoculação: 24 horas antes; simultânea a; e 24 horas após a inoculação de 10(4) esporos mL-1 de B. cinerea. A incidência, a percentagem de área de discos colonizados e a esporulação de B. cinerea foram avaliadas por meio de diagrama de notas, a partir do quarto dia após a inoculação. Todas as concentrações da levedura reduziram a esporulação de B. cinerea nos discos de folha, em comparação à testemunha, e a concentração de 10(7) esporos mL-1 foi a mais eficiente.

Vaporização de ácido acético para o controle pós-colheita de botrytis cinerea em uva 'Itália'

Visando a avaliar o efeito do vapor de ácido acético (AA) como medida alternativa no controle pós-colheita do mofo-cinzento (Botrytis cinerea) em uva 'Itália', foram conduzidos dois ensaios in vivo onde se avaliou o efeito direto e indireto do AA através do tratamento dos cachos antes e após a inoculação do patógeno, sendo: 1) cachos de uva foram inoculados e, após 4 h, vaporizados com AA (0,0; 0,25; 0,5; 1,0 ou 2,0 mL 100 L-1 vol. de câmara) em tambores herméticos (200 L), a 25±1 ºC / 70-80 % UR, por 30 min; 2) cachos foram, ou não, vaporizados com AA (1 mL 100 L-1 vol. de câmara) e, após 24; 48; 72 ou 96 h, inoculados com B. cinerea. Para inoculação, em cada cacho, 10 por tratamento, foram feridas 10 bagas, fazendo-se um furo por baga de ±2 mm de profundidade, procedendo-se, em seguida, aspersão de suspensão de esporos (±105 conídios mL-1). Após os tratamentos, os cachos foram mantidos a 25±1 ºC / 80-90 % UR e avaliados diariamente quanto à incidência e severidade da podridão. O efeito in vitro do vapor de AA no controle do patógeno foi avaliado com o intuito de verificar se tal agente estaria exercendo efeito direto sobre o crescimento micelial e a germinação de conídios de B. cinerea. Nos ensaios in vivo, o vapor de AA controlou a podridão de B. cinerea em uva 'Itália', nos cachos inoculados antes ou após o tratamento com AA, sendo as uvas inoculadas 48 h após o tratamento, as que apresentaram menor índice de doença. In vitro, o AA exerce efeito direto sobre o crescimento micelial e a germinação de conídios de B. cinerea.

Survival of Botrytis cinerea as mycelium in rose crop debris and as sclerotia in soil

Botrytis blight caused by Botrytis cinerea is an important disease of rose (Rosa hybrida) grown in greenhouses in Brazil. As little is known regarding the disease epidemiology under greenhouse conditions, pathogen survival in crop debris and as sclerotia was evaluated. Polyethylene bags with petals, leaves, or stem sections artificially infected with B. cinerea were mixed with crop debris in rose beds, in a commercial plastic greenhouse. High percentage of plant parts with sporulation was detected until 60 days, then sporulation decreased on petals after 120 days, and sharply decreased on stems or leaves after 90 days. Sporulation on petals continued for 360 days, but was not observed on stems after 150 days or leaves after 240 days. Although the fungus survived longer on petals, stems and leaves are also important inoculum sources because high amounts of both are deposited on beds during cultivation. Survival of sclerotia produced on PDA was also quantified. Sclerotia germination was greater than 75% in the initial 210 days and 50% until 360 days. Sclerotia weight gradually declined but they remained viable for 360 days. Sclerotia were produced on the buried petals, mainly after 90 days of burial, but not on leaves or stems. Germination of these sclerotia gradually decreased after 120 days, but lasted until 360 days. Higher weight loss and lower viability were observed on sclerotia produced on petals than on sclerotia produced in vitro

Avaliação de quitosana, aplicada em pós-colheita, na proteção de uva 'Itália' contra Botrytis cinerea

Perdas significativas ocorrem durante o armazenamento e a comercialização de uvas de mesa devido, principalmente, à ocorrência do mofo cinzento (Botrytis cinerea Pers.:Fr.) e, para o controle de patógenos emprega-se, geralmente, o dióxido de enxofre (SO2). Diante da restrição crescente ao uso de produtos químicos em pós-colheita, tem ocorrido considerável interesse em métodos alternativos de controle. Este trabalho teve como principal objetivo avaliar os efeitos da quitosana, na proteção pós-colheita de uva 'Itália' contra B. cinerea. In vivo, avaliou-se o efeito direto e indireto da quitosana pelo tratamento dos cachos de uva, antes e após a inoculação com o patógeno. Utilizou-se quitosana nas concentrações de 0,00; 0,25; 0,50; 1,00; 1,50 e 2,00 % (v/v). Para inoculação, em 10 bagas de cada cacho de uva foram feitos ferimentos de ±2 mm de profundidade, procedendo-se em seguida, a aspersão da suspensão de conídios (±10(5) conídios.mL-1) de B. cinerea. Após os tratamentos, os cachos foram mantidos a 25±1 °C / 80-90 % UR e avaliados diariamente quanto à incidência e severidade da podridão. Avaliações in vitro do efeito do produto sobre o patógeno também foram realizadas analisando-se o crescimento micelial e a germinação dos conídios de B. cinerea. A solução de quitosana, nas concentrações de 1,5 e 2,0 % (v/v), quando empregada após a inoculação com B cinerea, reduziu significativamente o índice de doença no entanto, quando os cachos foram tratados antes da inoculação, não houve efeito significativo do tratamento sobre o desenvolvimento da doença. Nos ensaios in vitro, a solução de quitosana, nas maiores concentrações, suprimiu o crescimento micelial do patógeno e retardou a germinação dos conídios.

Efeito de variáveis ambientais, épocas e métodos de plantio na intensidade da seca da haste (Botrytis cinerea) em Hibiscus sabdariffa

O presente estudo objetivou avaliar o efeito da temperatura (15, 20, 25 e 30ºC), do período de molhamento foliar (0, 6, 12 e 24 h), de épocas (setembro, outubro, novembro e dezembro) e métodos de plantio (semeadura direta e transplantio de mudas), na intensidade da seca da haste (Botrytis cinerea) do hibisco (Hibiscus sabdariffa). As variáveis ambientais foram avaliadas em condições controladas com inoculação artificial e as épocas e métodos de plantio foram avaliados em condições de infecção natural em campo. Os dados de frequência de infecção analisados, como área abaixo da curva de progresso da frequência de infecção (AACPF) e comprimento de lesões relacionados às variáveis ambientais, foram submetidos à análise de variância e regressão e, em seguida, plotadas as superfícies de resposta. Os dados de incidência (AACPI) relacionados às épocas e métodos de plantio foram submetidos à análise de variância, utilizando-se o programa estatístico Sisvarâ/UFLA. A interação da temperatura e da duração do período de molhamento foliar influenciou a frequência de infecção e o comprimento de lesões da seca da haste. Houve aumento na frequência de infecção e no comprimento de lesões com o incremento do período de molhamento foliar e redução da temperatura. As lesões apresentaram maior tamanho na temperatura de 15ºC e 24 horas de molhamento foliar. Na ausência de molhamento foliar houve manifestação de sintomas somente a 15ºC. A 30ºC houve dependência de maior período de molhamento foliar para a manifestação de sintomas. Houve interação significativa de métodos e épocas de plantio na incidência da doença. Constatou-se menor incidência da seca da haste em transplantio de mudas comparado à semeadura direta em todas as épocas de plantio. Verificou-se aumento da incidência proporcionado pelo atraso na época de plantio nos dois métodos. Registrou-se uma relação direta entre queda de temperatura e aumento da incidência da seca da haste.

Progresso da seca da haste (Botrytis cinerea) do hibisco (Hibiscus sabdariffa) em quatro épocas e dois métodos de plantio

O progresso da seca da haste em hibisco foi estudado em quatro épocas e dois métodos de plantio. O delineamento experimental foi em blocos ao acaso, com quatro repetições e os oito tratamentos foram dispostos em esquema fatorial 4 (épocas de plantio) x 2 (métodos de plantio), sendo: semeadura direta em 05/02/03, 06/03/03, 05/04/03 e 15/05/03; transplantio de mudas em 24/12/02, 25/01/03, 24/02/03 e 25/03/03. Imediatamente após o surgimento dos sintomas, avaliou-se a doença a cada 10 dias até o final do ciclo, aos 205 dias, calculando-se a porcentagem de hastes infectadas por planta. Calculou-se a área abaixo da curva de progresso para a incidência (AACPI). As curvas de progresso da doença dos tratamentos foram submetidas ao ajuste dos modelos linear, exponencial, monomolecular, logístico e Gompertz. Houve interação significativa de métodos e épocas de plantio na incidência da doença. Constatou-se menor incidência da seca da haste em transplantio de mudas comparado à semeadura direta em todas as épocas de plantio. Verificou-se aumento da incidência proporcionado pelo atraso na época de plantio nos dois métodos. O modelo exponencial foi o que melhor descreveu o comportamento da doença em todos os tratamentos. As diferenças estatísticas entre os tratamentos, considerando a taxa de progresso, não refletiram a intensidade da doença no campo. Na mesma época de plantio, as quantidades inicial e máxima da doença, observadas na semeadura direta, foram superiores aos tratamentos referentes ao transplantio de mudas, coerentes com os valores da AACPI. Registrou-se uma relação direta entre queda de temperatura e aumento da incidência da seca da haste.

Key factors to inoculate Botrytis cinerea in tomato plants

Studies addressing the biological control of Botrytis cinerea have been unsuccessful because of fails in inoculating tomato plants with the pathogen. With the aim of establishing a methodology for inoculation into stems, experiments were designed to assess: i. the aggressiveness of pathogen isolates; ii. the age at which tomato plants should be inoculated; iii. the susceptibility of tissues at different stem heights; iv. the need for a moist chamber after inoculation; and v. the effectiveness of gelatin regarding inoculum adhesion. Infection with an isolate from tomato plants that was previously inoculated into petioles and then re-isolated was successful. An isolate from strawberry plants was also aggressive, although less than that from tomato plants. Tomato plants close to flowering, at 65 days after sowing, and younger, middle and apical stem portions were more susceptible. There was positive correlation between lesion length and sporulation and between lesion length and broken stems. Lesion length and the percentage of sporulation sites were reduced by using a moist chamber and were not affected by adding gelatin to the inoculum suspension. This methodology has been adopted in studies of B. cinerea in tomato plants showing reproducible results. The obtained results may assist researchers who study the gray mold.

Efeito de Botrytis cinerea na composição do vinho Gewürztraminer

Com o objetivo de estudar o efeito da podridão cinzenta da uva, causada por Botrytis cinerea na composição do vinho Gewürztraminer (Vitis vinifera L.), foram analisados vinhos obtidos a partir de uvas com 0; 2,5; 5; 10; 15; e 20% em peso de podridão cinzenta. O experimento consistiu de seis tratamentos e quatro repetições. As variáveis avaliadas foram densidade, álcool, acidez total e volátil, extrato seco, extrato seco reduzido, açúcares redutores, relação álcool em peso/extrato seco reduzido, cinzas, alcalinidade das cinzas, índice de cor (I 420), polifenóis totais (I 280), N total, glicerol, ácido glicônico, minerais (P, K, Ca, Mg, Na, Mn, Fe, Cu, Zn, Rb, Li), compostos voláteis (etanal, acetato de etila, metanol, 1-propanol, 2-metil-1-propanol, 2-metil-1-butanol e 3-metil-1-butanol). A análise de regressão polinomial que avaliou o efeito da podridão cinzenta da uva na composição físico-química do vinho Gewürztraminer, mostrou que houve efeito linear significativo e positivo em relação à densidade, acidez total e volátil, extrato seco, extrato seco reduzido, açúcares redutores, cinzas, alcalinidade das cinzas, índice de cor (I 420), polifenóis totais (I 280), ácido glicônico, P, K, Ca, Mn, Fe, Rb, etanal e acetato de etila; efeito linear significativo e negativo nas variáveis álcool, relação álcool em peso/extrato seco reduzido, 2metil-1-butanol e 3-metil-1-butanol; efeito quadrático sobre N total, glicerol e metanol; e efeito cúbico sobre Mg, Zn, Li e 1-propanol. Não houve efeito significativo nas variáveis Na, Cu e 2-metil-1-propanol.

Characterization and protein fingerprinting of Botrytis cinerea isolates /

Botrytis cinerea isolates collected from Niagara region were treated with different concentrations of the fiingicide, iprodione to test their sensitivity to this fungicide. These Botrytis cinerea isolates were divided into two groups according to their sensitivity to iprodione. Those isolates whose growth was inhibited by iprodione at concentrations < 2|i,g/nil were classified as sensitive isolates. Isolates that were able to show considerable growth at 2|j,g/ml iprodione were classified as resistant isolates. Resistant and sensitive isolates were compared for their morphological and growth characteristics, conidial germination, virulence on grape berries and protein banding profiles. The fungicide iprodione at a concentration of 2|xg/nil inhibited mycelial growth, sporulation and conidial germination of sensitive isolates but not those of resistant isolates. The inhibitory effect of the fungicide was greater on mycelial growth than on conidia germination of the sensitive isolates. Sensitive isolates produced no sclerotia whereas resistant isolates produced large number of sclerotia. The fungicide iprodione affected sclerotial production in the resistant isolates. The number of sclerotia was decreased by the increase of iprodione in the medium. Sporulation of resistant isolates was improved significantly in the presence of iprodione. The resistant isolates were as virulent as the sensitive isolates on grape berries. The sensitive and resistant isolates showed similar protein banding profiles in the absence of iprodione in polyacrylamide gel electrophoresis studies. Similar protein profiles were also observed when these isolates were grown in the presence of low iprodione concentration (0.5|ig/nil). However, in the presence of concentration (0.5|ig/nil). However, in the presence of iprodione at concentration of 5|Xg/nil, one protein band with approximate molecular weight of 83 KDa was present in the growing resistant isolates (and the controls) but was missing in the inhibited sensitive isolates.

Alicaligenes faecalis : identification and study of its antagonistic properties against Botrytis cinerea

A Gram negative aerobic flagellated bacterium with fungal growth inhibitory properties was isolated from a culture of Trichoderma harzianum. According to its cultural characteristics and biochemical properties it was identified as a strain of Alcaligenes (aeca/is Castellani and Chalmers. Antisera prepared in Balbc mice injected with live and heat-killed bacterial cells gave strong reactions with the homologous immunogen and with ATCC 15554, the type strain of A. taeca/is, but not with Escherichia coli or Enterobacter aerogens in immunoprecipitation and dot immunobinding assays. Growth of Botrytis cinerea Pers. and several other fungi was significantly affected when co-cultured with A. taeca/is on solid media. Its detrimental effect on germination and growth of B. cinerea has been found to be associated with antifungal substances produced by the bacterium and released into the growth medium. A biotest for the antibiotic substances, based on their inhibitory effect on germination of B. cinerea conidia, was developed. This biotest was used to study the properties of these substances, the conditions in which they are produced, and to monitor the steps of their separation during extraction procedures. It has been found that at least two substances could be involved in the antagonistic interaction. One of these is a basic volatile substance and has been identified as ammonia. The other substance is a nonvolatile, dialysable, heat stable, polar compound released into the growth medium. After separation of growth medium samples by Sephadex G-10 column chromatography a single peak with a molecular weight below 700 Daltons exhibited inhibitory activity. From its behaviour in electrophoretic separation in agarose gels it seems that this is a neutral or slightly positively charged.