The significance of volatile antifungal metabolites produced by trichomerma harzianum biotype Th4, in green-mould disease of commercial mushroom crops /

The aggressive mushroom competitor, Trichoderma harzianum biotype Th4, produces volatile antifungal secondary metabolites both in culture and during the disease cycle in compost. Th4 cultures produced one such compound only when cultured in the presence of Agaricus bisporus mycelium or liquid medium made from compost colonised with A. bisporus. This compound has TLC and UVabsorption and characteristics indicating that it belongs to a class of pyrone antibiotics characterised from other T. harzianum biotypes. UV absorption spectra indicated this compound was not 6-pentyl-2H-pyran-one (6PAP), the volatile antifungal metabolite widely described in Th1. Furthermore, this compound was not produced by Th1 under any culture conditions. Mycelial growth of A. bisporus, Botrytis cinerea and Sclerotium cepivorum was inhibited in the presence of this compound through volatility , diffusion and direct application. This indicates that Th4 produces novel, volatile, antifungal metabolites in the presence of A. bisporus that are likely involved in green mould disease of mushroom crops.

Characterization of the chitinolytic system during the mycoparasitic interaction between Trichoderma aggressivum f. aggressivum and different host strains of Agaricus bisporus /

Green mould is a serious disease of commercially grown mushrooms, the causal agent being attributed to the filamentous soil fungus Triclzodenna aggressivum f. aggressivu11l and T. aggressivum f. ellropaellm. Found worldwide, and capable of devastating crops, this disease has caused millions of dollars in lost revenue within the mushroom industry. One mechanism used by TricllOdenlla spp. in the antagonism of other fungi, is the secretion of lytic enzymes such as chitinases, which actively degrade a host's cell wall. Therefore, the intent of this study was to examine the production of chitinase enzymes during the host-parasite interaction of Agaricus bisporus (commercial mushroom) and Triclzodemza aggressivum, focusing specifically on chitinase involvement in the differential resistance of white, off-white, and brown commercial mushroom strains. Chitinases isolated from cultures of A. bisporus and T. aggressivu11l grown together and separately, were identified following native PAGE, and analysis of fluorescence based on specific enzymatic cleavage of 4-methylumbelliferyl glucoside substrates. Results indicate that the interaction between T. aggressivulll and A. bisporus involves a complex enzyme battle. It was determined that T. aggressivum produces a number of chitinases that appear to correlate to those isolated in previous studies using biocontrol strains of T. Izarziallilm. A 122 kDa N-acetylglucosaminidase of T. aggressivu11l revealed the highest and most variable activity, and is therefore believed to be an important predictor of antifungal activity. Furthermore, results indicate that brown strain resistance of mushrooms may be related to high levels of a 96 kDa N-acetylglucosaminidase, which showed elevated activity in both solitary and dual cultures with T. aggressivum. Overall, each host-parasite combination produced unique enzyme profiles, with the majority of the differences seen between day 0 and day 6 for the extracellular chitinases. Therefore, it was concluded that the antagonistic behaviour of T. aggressivli1ll does not involve a typical response, always producing the same types and levels of enzymes, but that mycoparasitism, specifically in the form of chitinase production, may be induced and regulated based on the host presented.

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.

Investigations on the host-parasite interface of a mycoparasite system /

The cell wall composition of Choanephora cucur - bitarum and the host-parasite interface, after infection with Piptocephalis virginiana , were examined in detail. The cell walls of C_. cucurbitarum were determined to be composed of chitin (17%), chitosan (28.4%), neutral sugars (7.2%),uronic acid (2.4%), proteins (8.2%) and lipids (13.8%). The structure of hyphal walls investigated by electron microscopy of shadowed replicas before and after alkali-acid hydrolysis, showed two distinct regions: microfibrillar and amorphous. The microfibrils which were composed of mainly chitin, were organized into two distinct layers: an outer, thicker layer of randomly orientated microfibrils and an inner, thin layer of parallel microfibrils.Electronmicrographs of the host-parasite interface of C_. cucurbitarum and the mycoparasite , P_. virginiana , 30 h following inoculation, showed that the sheath zone has a similar electron density to that of the host cell wall. The sheath was not present around the young (18 h old) haustorium. High-resolution autoradiographs of infected host hyphae showed that radioactive N-acetyl-D-glucosamine , a precursor of chitin, was incorporated preferentially in the host cell wall and sheath zone. Cell fractionation of label fed hyphae showed that 84% of the label was present in the cell wall and specifically in the chitin portion of the wall. The antifungal antibiotic, Polyoxin D, a specific inhibitor of the enzyme, chitin synthetase, suppressed the incorporation of the label in the cell wall and sheath zone and resulted in a decrease in electron density of the developing sheath. The significance of these results is discussed in the light of host resistance.