The symbiotic relationship between termites and Termitomyces fungi, which allows the termite to digest cellulose-rich food sources, is poorly understood. In this study, in vitro mixed symbiotic relationships between Termitomyces clypeatus and fungi isolated from individual fungus-comb communities using a culture-dependent method were analyzed. Twenty-day-old stalk cultures of three T. clypeatus isolates were co-cultured with cellulase-producing fungi on potato dextrose agar. The high cellulase-producing fungal isolate no. 18, which showed 99 % ITS sequence identity to Sordariomycetes endophyte isolate 2171 (EU687039), increased growth of T. clypeatus 18/50 by 85.7 %. The high xylanase-producing isolate no. 13, which showed 88 % ITS sequence identity to Arthrinium sacchari isolate L06 (HQ115662), stimulated T. clypeatus 18/50 growth by 58.6 %. The high cellulase- and xylanase-producing isolate no. 50, which showed 90 % ITS sequence identity to the fungal endophyte isolate 2196 (EU687056), improved T. clypeatus 18/50 growth by 45.7 %. A Gigantropanus sp. promoted the growth of T. clypeatus 18/50 and 20/50 by 45.7 and 44.1 %, respectively, and that of T. clypeatus 19/50 by 10.6 %. These results indicated the most beneficial potential partnership of T. clypeatus might involve cellulase-producing fungi isolated from the same ecological niche. The Gigantropanus sp. is a potential partner of T. clypeatus but is likely to be less common than cellulase-producing fungi isolated from fungus combs owing to the lower host specificity of the Gigantropanus sp. This study provides an interesting method to culture Termitomyces using an in vitro mixed culture method for production of Termitomyces fruiting bodies in the future.
The Type A isolate of Colletotrichum gloeosporioides from Stylosanthes hamata and an isolate from Aeschynomenefalcata had relatively wide host ranges, causing disease on 10 and 11 respectively of the 22 plant species tested. Seven plant species were common hosts to the two strains. The Type B isolate from Stylosanthes guianensis had a relatively narrow host range, causing severe disease on S. guianensis only and slight disease on Desmodium barbatum only. Because of this, a special form is proposed for the Type B isolate, namely C. gloeosporioides f. sp. guianensis, to indicate its specificity towards Stylosanthes guianensis. The histopathology at the light microscope level of the non-host resistance of S. scabra to C. Gloeosporioides f. sp. guianensis was studied both on whole leaves that had been cleared and stained, and in transverse sections. The compatible interaction between S. guianensis and C. gloeosporioides f. sp. guianensis was also studied in parallel as a control. Quantitative whole leaf studies showed that the pre-penetration processes were similar for the two interactions, with higher levels of germination occurring on the non-host (35%) than on the host (27%). Of 2250 appressoria examined for evidence of penetration in whole leaves for each of the two interactions at 72 h after inoculation, 6.6% had penetrated for the compatible interaction and in all cases only direct penetration was observed. For the interaction with the non-host (S. scabra) only one penetration was observed, and this appeared to occur through a stomate. The studies on transverse leaf sections showed that in the non-host interaction, penetration pegs, if produced, remained localised in the cuticle and were surrounded by a densely staining reaction matrix. In the compatible interactions, the fungus produced a vesicle in the cuticle by 24 h. A dense reaction matrix was often observed in the cuticle beneath the appressorium but penetration of the host epidermal cell, followed by subcuticular, intercellular and intracellular growth, had occurred by 72 h. Cell collapse was not evident after this period, suggesting the fungus was biotrophic for the first 72 h of the interaction.
The extent of variation for host disease reaction and pathogen virulence was studied in naturalized populations of Stylosanthes hurnilis and Colletotrichurn gloeosporioides, the causal fungus of an anthracnose disease of Stylosanthes spp. Diseased plants (S0) were collected from the field at three sites (Townsville, Wrotham Park and Niall) in North Queensland, and first generation selfed (S1) progenies (host-lines) and single spore fungal cultures were grown for each of the collections made. Within a site, all host-lines were inoculated with each fungal isolate from that site, and a fourth experiment was conducted with representative host-pathogen combinations from each site. Sufficient seed was obtained to allow testing of 12, 10 and 8 collections from Niall, Wrotham Park and Townsville respectively. Significant variation (P < 0.01) between disease severity values for host-line means, fungal isolate means and host-line/fungal isolate interactions was found in all four experiments. Differences between fungal isolate means were the main source of variation in three of the four experiments. Both the differences in virulence within the pathogen population and the differences in resistance of the hostlines appeared to be quantitatively inherited. One host-line from Wrotham Park was significantly more resistant than the susceptible check, cv. Paterson, in two replicated experiments indicating that selection for some improvement in resistance within the naturalized populations should be possible. However, none of the host-lines from the Townsville and Niall sites were significantly more resistant than Paterson, suggesting that little natural selection for resistance has occurred within the naturalized host populations over the 10 years following the first outbreak of the disease in northern Australia.
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