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.

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.

Immuno-cytochemical localization of glycoproteins involved in recognition and attachment in a mycoparatism

Polyclonal antibodies prepared against the two glycoproteins (Mr 100 and 85 kDa) involved in recognition and attachment of the mycoparasite, Piptocephalis virginiana, to its hosts, Mortierella pusilla and Phascolomyces articulosus, susceptible and resistant, respectively, were employed to localize the antigens at their cell surfaces. Indirect immunocytochemical technique using secondary antibodies labelled with either FITC or gold particles as probes, were used. FITC-Iabelled antibodies revealed a discontinous pattern of fluorescence on the hyphae of MortlerelLa pusilla and no fluorescence on the hyphae of Phascolomyces articulosus. Intensity of fluorescence was high in the germinating spores of both the fungi. Fluoresence could be observed on P. articulosus hyphae pretreated with a commercial proteinase. Fluorescence was not observed on either hyphae or germinating spores of the nonhost M0 r tie re11 a ca ndelabrum and the mycoparasite P. virginiana. Antibodies labelled with gold conjugate showed a different pattern of antigen localization on the hyphal walls of the susceptible and resistant hosts. Patches of gold particles were observed allover the whole cell wall of the susceptible host but only on the inner cell wall layer of the resistant host. Cell wall fragments of the susceptible host but not those of the resistant host, previously incubated with the antibodies inhibited attachment of the mycoparasite. Implications of preferential localization of the antigen in the resistant host and its absence in the nonhost are described.

Involvement of fimbriae in host-mycoparasite recognition

Extracellular, non-flagellar appendages, termed fimbriae are widespread among fungi. Fungal fimbriae range in diameter from 6-10 nm and exhibit lengths of up to 30 ~m. Fungal fimbriae have been implicated in several functions: adhesion, conjugation and flocculation. A possible role of fimbriae in host-mycoparasite interactions was the focus of this study . Using electron microscopy, fimbriae were observed on the surfaces of Mortiere lla cande labrum, Mortie re lla pusi lla and Phascolomyces articulosus with diameter means of 9.1±0.4 nm, 9.4±0.5 nm and 8.6±0.6 nm, respectively, and lengths of up to 25 ~m. Fimbriae were not observed on the surface of the mycoparasite, Piptocephalis virginiana. Polyclonal antiserum (AU) prepared against the fimbrial protein of Ustilago violacea cross-reacted with 60 and 57 kDa M. candelabrum proteins. In addition, AU cross-reacted with 64 kDa proteins from both M. pusilla and P. articulosus. The proteins that cross-reacted with AU were electroeluted from polyacrylamide gels and were shown to subsequently form fibrils. The diameter means for the electroeluted fibrils were: for M. candelabrum 9.7±0.3 nm, M. pusilla 8.4±0.6 nm and P articulosus 9.2±0.5 nm. Finally, to ascertain the role of fimbriae in host-mycoparasite interactions, AU was incubated with P. virginiana and M. pusilla (mycoparasite/susceptible host) and with P. virginiana and P . articulosus (mycoparasite/ resistant host). It was observed that AU decreased significantly the level of contact between P. virginiana and M. pusilla and between P. virginiana and P. articulosus in comparison to prelmmune serum treatments. Thus, it was proposed that fimbriae might play recognition and attachment roles in early events of mycoparasitism.

Isolation and partial characterization of a complementary protein from the mycoparasite, Piptocephalis virginiana, which specifically binds to two glycoproteins b and c of the host cell surface

Presence of surface glycoprotein in Piptocephalis virginiana that recognizes the host glycoproteins band c, reported earlier from our laboratory, was detected by immunofluorescence microscopy. Germinated spores of P. virginiana treated with Mortierella pusilla cell wall protein extract, primary antibodies prepared against glycoproteins band c and FITC-goat anti-rabbit IgG conjugate showed fluorescence. This indicated that on the surfaces of the biotrophic mycoparasite P. virginiana , there might be a complementary molecule which recognizes the glycoproteins band c from M. pusilla. Immunobinding analysis identified a glycoprotein of Mr 100 kDa from the mycoparasite which binds with the host glycoproteins band c, separately as well as collectively. Purification of this glycoprotein was achieved by (i) 60% ammonium sulfate precipitation, (ii) followed by heat treatment, and (iii) Sephadex G-IOO gel filtration. The glycoprotein was isolated by preparative polyacrylamide gel electrophoresis by cutting and elution. The purity of the protein ·was ascertained by SDS-PAGE and Western blot analysis. Positive reaction to periodic acid-Schiff reagent revealed the glycoprotein nature of this 100 kDa protein. Mannose was identified as a major sugar component of this glycoprotein by using a BoehringerMannheim Glycan Differentiation Kit. Electrophoretically purified glycoprotein was used to raIse polyclonal antibody in rabbit. The specificity of the antibody was determined by dot-immunobinding test and western-blot analysis. Immunofluorescence mIcroscopy revealed surface localization of the protein on the germ tube of Piptocephalis virginiana. Fluorescence was also observed at the surfaceJ of the germinated spores and hyphae of the host, M. pusilla after treatment with complementary protein from P. virginiana, primary antibody prepared against the complementary protein and FITC-goat anti-rabbit IgG conjugate.

Diversity among bacteria causing blotch disease on the commercial mushroom, agaricus bisporus /

A total of 251 bacterial isolates were isolated from blotched mushroom samples obtained from various mushroom farms in Canada. Out of 251 stored isolates, 170 isolates were tested for pathogenicity on Agaricus bisporus through mushroom rapid pitting test with three distinct pathotypes observed: dark brown, brovm and yellow/yellow-brown blotch. Phenotypic analysis of 83 isolates showed two distinct proteinase K resistant peptide profiles. Profile group A isolates exhibited peptides with masses of 45, 18, 16 and 14 kDa and fiirther biochemical tests identified them as Pseudomonasfluorescens III and V. Profile group B isolates lacked the 16-kDa peptide and the blotch causing bacterial isolates of this group was identified as Serratia liquefaciens and Cedecea davisae. Comparative genetic analysis using Amplified Fragment Length Polymorphism (AFLP) on 50 Pseudomonas sp. isolates (Group A) showed that various blotch symptoms were caused by isolates distributed throughout the Pseudomonas sp. clusters with the exception of the Pseudomonas tolaasii group and one non-pathogenic Pseudomonas fluorescens cluster. These results show that seven distinct Pseudomonas sp. genotypes (genetic clusters) have the ability to cause various symptoms of blotch and that AFLP can discriminate blotch causing from non-blotch causing Pseudomonasfluorescens. Therefore, a complex of diverse bacterial organisms causes bacterial blotch disease

Isolation and analysis of a corprinus cinereus DNA fragment showing homology to a fimbrial cDNA from ustilago violacea

Surface proteinaceous fibrils, termed fimbriae, were first identified on gram negative bacteria in the 1940s. Fungal fimbriae, discovered some 25 years later, are found on members of all fungal classes. In the present study, polyclonal antiserum raised against the fimbrial proteins of U. vio/acea were used in order to identify antigenically related proteins from Coprinus cinereus and Schizophy//um commune. Two polypeptides with molecular masses of 37 and 39 kDa from C. cinereus were observed and confirm earlier results. A single previously unidentified 50 kDa polypeptide in S. commune crossreacted with the antiserum. The 50 kDa protein was found to consist of 3 isoforms with isoelectric points ranging from 5.6 to 5.8. A fimbrial cDNA derived from U. vio/acea was used to identify DNA restriction fragments from C. cinereus and S. commune showing homology to the fimbrial transcript of U. vio/acea. Heterologous hybridization with this cDNA was used in order to screen a C. cinereus genomic DNA library. A single clone, A2-3A, with a 14 kbp insert showed strong homology to the pfim3-1 cDNA. The region of homology, a 700 bp Xba I fragment, was subcloned into pUG19. This plasmid was refered to as pXX8. DNA sequence determinations of pXX8 and adjacent fragments from A2-3A suggested that the cloned DNA was a portion of the rONA repeat encoding the small subunit rRNA. DNA sequence analysis of pfim3-1 yielded an incomplete open reading frame. The predicted amino acid sequence codes for a 206 amino acid, 22 kDa polypeptide which contains a domain similar to a transmembrane domain from rat leukocyte antigen, GDS3. As well, an untranslated 576 nucleotide domain showed 81 % homology to pXX8 and 830/0 homology to the 188 rRNA sequence of Ustilago maydis. This sequence was found adjacent to a region of adenine-thymine base pairs presumed to represent the polyadenylation sequence of the fimbrial transcript. The size and extent of homology is sufficient to account for the hybridization of pfim3-1 to rDNA. It is suggested that this domain represents a completely novel regulatory domain within eukaryotes that may enable the observed rapid regeneration of fimbriae in U. violacea.

Characterization of black fly (Diptera: Simuliidae) silk proteins

Black fly (Simuliidae) silk is produced by the larvae and pharate pupae and is used for anchorage and cocoon production. There exists limited information on simuliid silks, including protein composition and genetic sequences encoding such proteins. The present study aimed to expand what is known about simuliid silks by examining the silks of several simuliid species and by making comparisons to the silk of non-biting midges (Chironomidae). Silk glands were dissected out of larval and pupal simuliids, and protein contents were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and visualized with silver stain. Protein contents were compared by mass in kilodaltons (kDa) between life stages and among species. Polymerase chain reaction (PCR) was used to expand upon known gene sequence information, and to determine the presence of genes homologous to chironomid silk. SDS-PAGE of cocoons revealed the presence of a 56 kDa and a 67 kDa protein. Silk gland contained as many as 28 different proteins ranging from 319 kDa to 8 kDa. Protein profiles vary among species, and group into large (>200), intermediate(>100), and small (<100) protein classes as is found in chironomids. It is likely that silk evolved in a common ancestor of simuliids and chironomids