D. F. Cameron

Variation in virulence and pathogenic specialization of Colletotrichum gloeosporioides isolates from Stylosanthes scabra cvv. Fitzroy and Seca.

A survey of anthracnose of Stylosanthes scabra cvv. Fitzroy and Seca, caused by Colletotrichurn gloeosponoides, at 31 sites in Queensland found a wide range of disease severity. Disease was present at each of the 26 sites surveyed for Fitzroy, and inoculation experiments with an isolate from each site indicated that the isolates varied markedly in their virulence on Fitzroy. Some of the variation in disease severity between sites could be explained by differences In virulence of the isolates. Disease was observed In only one of the five fields of Seca surveyed. When nine of the isolates from Fitzroy were tested on SIX genotypes of S. scabra, there was no evidence of pathogenic specialization, and all isolates were avirulent on Seca. The isolate from Seca, when tested on the same genotypes, was moderately virulent on Seca and highly virulent on Fitzroy, indicating further pathogenic specialization within the Type A forms of C. gloeosporioides in Australia. There was a direct relationship between mean linear growth rate of isolates from Fitzroy in pure culture, and virulence on Fitzroy. The recovery of highly virulent Isolates from pods of a Fitzroy seed crop is discussed in relation to the rapid spread of highly virulent physiologic races.

Biology of Colletotrichum gloeosporioides isolates from tropical pasture legumes

The relationships between isolates of Colletotrichum gloeosporioides from Stylosanthes guianensis (an anamorphic and a teleomorphic isolate), Aeschynomene falcata and Centrosema pubescens were investi- gated by comparing their morphology, temperature relations, symptomatology and pathogenicity. The Stylosanthes and Aeschynomene isolates caused more severe symptoms on the host from which they were isolated than on the other hosts. The Centrosema isolate caused moderate infection on Stylosanthes and mild infection on Centrosema. The anamorphic isolate from Stylosanthes produced leaf blight while the other isolates produced localised necrotic lesions. Conidia of the Centrosema isolate and the teleomorphic isolate from Stylosanthes were relatively uniform in size and shape and rounded at both ends. Both isolates produced fertile perithecia. Conidia of the Aeschynomene isolate and the anamorphic Stylosanthes isolate were more variable in shape and usually tapered at one end. The optimum temperature for mycelial growth of the anamorphic Stylosanthes isolate and the Aeschynomene isolate was 25°C, and for growth of the teleomorphic Stylosanthes isolate and the Centrosema isolate 27-29°C. Infection by the anamorphic and the teleomorphic Stylosanthes isolates proceeded in a similar fashion. Germinated spores and unmelanised appressoria were first observed at 6 h, melanised appressoria at 12 h and multiple appressoria and penetration at 24 h after inoculation. The anamorphic isolate produced significantly more successful penetrations than the teleomorphic isolate.

Components of quantitative resistance to anthracnose (Colletotrichum gloeosporioides) in tetraploid accessions of the pasture legume Stylosanthes hamata

Partial resistance to the anthracnose pathogen Colletotriclzum gloeosporioides was characterised in 6 tetraploid accessions and cv. Verano of the tropical pasture legume Stylosanthes hamata. Four components of resistance (incubation period, latent period, spore production, weighted infection type) were determined and compared with those of the susceptible S. scabra cv. Fitzroy. There were significant differences between the accessions for all resistance components. The mechanisms of resistance in the 3 most resistant accessions appeared to be different, with accession 55830 having a longer incubation period but higher sporulation per day than either 92715 or 92412. Spore production, weighted infection type, and latent period were significantly (P<0.05) correlated with resistance of adult plants in the field (ADPC). The weighted infection type was useful in discriminating between accessions that had similar disease severity scores. A quantitative relationship was established between 2 components, latent period and spore production per day, and the ADPC, using a multiple regression analysis (r2 = 0.9). If verified using a large number of accessions, such analysis may be used to obtain an index of resistance that may predict resistance levels in the field based on the components.

Host range of three strains of Colletotrichum gloeosporioides from tropical pasture legumes, and comparative histological studies of interactions between type B disease-producing strains and Stylosanthes scabra (non-host) and S. guianensis (Host)

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.

Influence of environmental factors on the development of the anthracnose diseases of Stylosanthes spp

The influence of temperature, period of leaf wetness after inoculation, and relative humidity on the development of the type A and type B diseases of Stylosanthes spp. caused by Colletotrichum gloeosporioides was studied in controlled environment cabinets. Severe disease of both types developed in plants incubated at temperatures of 20-30¦C, and given 24 h of leaf wetness after inoculation. Provided these conditions were imposed, high levels of disease developed following the leaf wetness period, irrespective of the relative humidity (40-50% or >95%). High levels of disease developed for both the type A and type B combinations at temperatures in the range 20-3OoC, but only the type A disease developed at >34¦C. Neither disease developed at constant temperatures of 15 or 37¦C. The use of these results in the development of a model to predict field infection is discussed.

Histopathology of compatible interactions involving biotypes of Colletotrichum gloeosporioides that cause anthracnose of Stylosanthes spp

The histopathology of compatible interactions between type A and type B Colletotrichum gloeosporioides and Stylosanthes scabra cv. Fitzroy and Stylosanthes guianensis cv. Endeavour was studied by light microscopy and scanning electron microscopy at various times after inoculation. Quantitative studies of germination and penetration processes with whole leaf mounts showed that the processes were similar for the two diseases but that unmelanized appressoria were produced more rapidly (3 h after inoculation) by the type A fungus on both hosts than by the type B fungus on Endeavour (6 h after inoculation). After 6 h for the type A fungus and 12 h for the type B fungus, the majority of appressoria were melanized. First evidence of penetration on the whole leaf mounts was observed 12 h after inoculation on all three interactions. Percentage penetration of the susceptible Endeavour by the type B fungus was significantly higher than that for the type A fungus on Fitzroy (susceptible) and Endeavour (intermediate). Subcuticular infection hyphae were present in transverse leaf sections of both diseases after 12 h and both intercellular and intracellular fungal invasion were observed after 24 h. Acewuli of the type A hngus had developed on Fitzroy by 96 h, whereas acervuli of the type B fungus had developed on Endeavour by 144 h. Mesophyll cells were particularly susceptible to fungal invasion and rapidly collapsed. Swelling of epidermal cell walls in response to invasion was also observed but this did not confer resistance.

Epidemiological studies on the anthracnose diseases of Stylosanthes spp. caused by Colletotrichum gloeosporioides in North Queensland and pathogenic specialization within the natural fungal populations

Field experiments were conducted during three wet seasons (1982-84) on two sites in north Queensland to examine the natural occurrence of anthracnose caused by Collectotrichum gloeosporioides on six Stylosanthes spp. The disease incidence varied from moderate to high in a number of accessions, while others showed good field resistance for the duration of the experiment. Anthracnose severity was similar at both sites, although total rainfall each season was c. four times higher at one site than at the other. In some cases disease severity was higher at the drier site. It was difficult to determine a relationship between the environmental parameters measured (rainfall, temperature and relative humidity) and disease incidence and severity. Disease severity patterns varied between accessions at the same site, indicating a response to a factor or factors not associated with the measured weather parameters. There were seven pathogenicity groups, based on host specificities in glasshouse studies, identified within the Type A fungal population and a further seven groups within the Type B population. This indicates an increase in the number of fungal groups reported in Australia, and suggests that a stepwise increase in specificity may be occurring within the fungal population.

Histopathology of compatible and incompatible interactions between Colletotrichum gloeosporioides and Stylosanthes scabra

The histopathology of interactions between five genetically defined cultivars and accessions of Stylosanthes scabra and three pathotypes of Colletotrichum gloeosporioides was studied qualitatively and quantitatively. In all interactions studied prepenetration processes were generally similar, leading to the formation of predominantly sessile melanized appressoria. Penetration occurred only from melanized appressoria, and in both compatible and incompatible interactions. There were generally significantly higher penetration levels for the compatible interactions. Differences in the extent of fungal growth in the cells of compatible and incompatible interactions were apparent by 48 h after inoculation. In the incompatible interactions, colony areas did not increase after 48 h, and the fungus rarely penetrated more than two cells. Cell degeneration was not evident with light microscopy in the first 48 h, even though up to six cells were penetrated by this time in susceptible hosts. Collapse of epidermal and mesophyll cells in susceptible hosts was, however, evident by 72 h.

The influence of mixtures of Stylosanthes spp. accessions on the occurrence of anthracnose caused by Colletotrichum gloeosporioides

The effectiveness of using accession mixtures of Stylosanthes spp. to manage anthracnose (Colletotrichum gloeosporioides) in pastures in northern Australia was examined during three consecutive years. Two mixtures containing six accessions were compared with the components grown as pure stands. No significant differences in anthracnose incidence (proportion of infected plants/plot) were indicated between the two mixtures and the mean incidence of their respective components grown in pure swards. Areas under the disease progress curves for the accessions were not significantly different between pure and mixed stands of the cultivars other than Seca and Verano. Resistant cultivar Seca developed more disease in a mixture than in a pure stand, and moderately resistant Verano had less disease in a mixture than in a pure stand. In the short term, no apparent anthracnose control advantage is achieved in highly susceptible accessions of Stylosanthes spp. when they are included in mixtures with less susceptible accessions. Long term studies involving grazing animals are necessary to adequately evaluate control of this disease through the use of mixtures.

Glasshouse and field evaluation of quantitative resistance to Colletotrichum gloeosporioides in tetraploid accessions of Stylosanthes hamata

A total of 20 field isolates of Collectotrichum gloeosporioides from Stylosanthes hamata was tested for pathogenicity and differential virulence in glasshouse inoculation experiments. No pathogenic specialization towards S. hamata was noted, but all three major races of the pathogen causing Type A disease were detected using the host differential set of one S. viscose and four S. scabra accessions. There was also little evidence of pathogenic specialization to S. hamata when 37 accessions were inoculated with three isolates representing the three main Type A races on S. scabra and a field isolate from S. hamata cv. Verano. The resistance of 38 accessions of S. hamata, including cvv. Verano and Amiga, was evaluated by inoculating field plots with a mixture of four isolates of C. gloeosporioides. Anthracnose progress was monitored by regular disease assessments and accessions were compared using the terminal disease severity, the rate parameter of logistic and linear models of disease progress and the area under the disease progress curve (ADPC). Accessions showed a continuous range from moderate to high resistance, with Verano and Amiga taking intermediate values. Spearman rank correlations among ADPC, terminal severity in the field, apparent infection rate and the severity in glasshouse tests were generally significant. Several resistant accessions were identified with potential for use in a breeding program to improve the anthracnose resistance of cvv. Verano and Amiga.