Isolation and characterization of polymorphic microsatellite markers for Alternaria alternata

Alternaria alternata is of major significance as a food and feed contaminant and is able to produce a range of mycotoxins that may elicit adverse effects in both animals and humans. We describe the isolation and characterization of five microsatellite markers for studying A. alternata. Marker polymorphism was screened in 64 isolates of A. alternata. The number of alleles per locus ranged from eight to 24, and allelic diversity ranged from 0.425 to 0.882. These markers will be useful in the study of relationships and population genetics amongst isolates of A. alternata.     

Cross-pathogenicity of Rhizoctonia solani strains on pasture legumes in pasture-crop rotations

In Australia, in the past, pasture legumes were rotated mainly with cereals, but increasingly these rotations now involve pasture legumes with a wider range of crops, including legumes. This increasing frequency of the leguminous host in the rotation system may be associated with increased root rots in legumes in the current pasture-crop rotations. The primary aim of this study was to see whether the pathogenicity on pasture legumes of strains of Rhizoctonia solani sourced from lupins and cereals (common crops in rotation with pastures) is associated with increased incidence of root rots in pasture legumes in the disease conducive sandy soils of the Mediterranean regions of southern Australia. The second aim was to determine sources of resistance among newly introduced pasture legumes to R. solani strains originating from rotational crops as this would reduce the impact of disease in the pasture phase. Fifteen pasture legume genotypes were assessed for their resistance/susceptibility to five different zymogram groups (ZG) of the root rot pathogen R. solani under glasshouse conditions. Of the R. solani groups tested, ZG1–5 and ZG1–4 (both known to be pathogenic on cereals and legumes) overall, caused the most severe root disease across the genotypes tested, significantly more than ZG6 (known to be pathogenic on legumes), in turn significantly >ZG4 (known to be pathogenic on legumes) which in turn was >ZG11 (known to be pathogenic on legumes including tropical species). Overall, Ornithopus sativus Brot. cvs Cadiz and Margurita, Trifolium michelianum Savi. cvs Paradana and Frontier and T. purpureum Loisel. cv. Electro showed a significant level of resistance to root rot caused by R. solani ZG11 (root disease scores ≤1.2 on a 1–3 scale where 3 = maximum disease severity) while O. sativus cvs Cadiz and Erica showed a significant level of resistance to root rot caused by R. solani ZG4 (scores ≤1.2). O. compressus L. cvs Charano and Frontier, O. sativus cv. Erica, and T. purpureum cv. Electro showed some useful resistance to root rot caused by R. solani ZG6 (scores ≤1.8). This is the first time that cvs Cadiz, Electro, Frontier, Margurita and Paradana have been recognised for their levels of resistance to root rot caused by R. solani ZG11; and similarly for cvs Cadiz and Erica against ZG4; and for cvs Charano, Erica, and Electro against ZG6. These genotypes with resistance may also serve as useful sources of resistance in pasture legume breeding programs and also could potentially be exploited directly into areas where other rotation crops are affected by these R. solani strains. None of the tested genotypes showed useful resistance to R. solani ZG1–4 (scores ≥2.0) or ZG1–5 (scores ≥2.5). This study demonstrates the relative potential of the various R. solani ZG strains, and particularly ZG1–4, ZG1–5, ZG4 and ZG6 to attack legume pastures and pose a significant threat to non-pasture crop species susceptible to these strains grown in rotation with these pasture legumes. Significantly, the cross-pathogenicity of these strains could result in the continuous build-up of inoculum of these strains that may seriously affect the productivity eventually of legumes in all rotations. In particular, when choosing pasture legumes as rotation crops, caution needs to be exercised so that the cultivars deployed are those with the best resistance to the R. solani ZGs most likely to be prevalent at the location.     

An extracellular enzyme from Fusarium solani f. sp. phaseoli which catalyses hydration of the isoflavonoid phytoalexin, phaseollidin

Among the antimicrobial phytoalexins produced by Phaseolus vulgaris (French bean) are the prenylated isoflavonoids kievitone and phaseollidin. Two enzyme activities, kievitone hydratase and phaseollidin hydratase, occur in culture filtrates of the bean pathogen, Fusarium solani f. sp. phaseoli, and catalyse similar hydration reactions on the dimethylallyl moieties of the phytoalexins. The enzymes nearly co-purified during hydroxyapatite chromatography followed by preparative native gel electrophoresis. Eluates from successive slices taken from the native gel were assayed for both activities. Although they were not completely separated in the native gel, the activity profiles indicated that the two activities were distinct. The Km of phaseollidin hydratase for phaseollidin was approximately 7 microM.     

Identification of two Bacillus amyloliquefaciens strains with high suppression to the key fruit pathogens of Chinese jujube

Two bacterial strains (ZJ01 and ZJ02) isolated from the phyllosphere of Chinese jujube were tested for antifungal activities and morphological mechanisms against Phoma destructiva, Alternaria alternata and Fusicoccum spp., three key pathogens of jujube fruits. Based on their physiological characteristics and 16S rDNA gene sequences, both strains were identified as Bacillus amyloliquefaciens. The disease incidence of jujube fruits sprayed with the fermentation liquor of ZJ01 and ZJ02 on the puncture wounds before inoculation with the spores of those three key pathogenic fungi was 10% or 66.7%, significantly lower than that of Nutrient Broth medium treatment (100%). These results suggest that strains ZJ01 may be a potential biological control agent for Chinese jujube fruit diseases. The antagonistic bacteria could cause five kinds of hyphal alterations of the causal agents, the maximum number ever observed. Of them, hyphal swelling and excessive branching were the dominant phenomenon, the other three varied with the pathogens. The diverse morphological malformation might be helpful to understand the broad antifungal activity of Bacillus spp..     

Hydroponic Seedling Bioassay for the Bioherbicides Colletotrichum truncatum and Alternaria cassiae

A rapid bioassay was developed to measure the bioherbicidal efficacy of spore preparations of the pathogens Colletotrichum truncatum (Schwein.) Andrus and W. D. Moore and Alternaria cassiae Jurair and Khan on hemp sesbania (Sesbania exaltata) and sicklepod (Cassia obtusifolia), respectively. The system uses 4-day-old dark-grown seedlings (grown hydroponically in paper towel cylinders) which were sprayed with spore suspensions. Shoot lengths were monitored non-destructively, and recorded over time under conditions of dark growth, 90-100% relative humidity and 25 C. Shoot growth inhibition and stem collapse (mortality) were directly related to the spore concentration applied. Generally, at 10 3 - 10 4 spores ml-1, these pathogens caused significant shoot growth inhibition within 25-30 h and seedling death within 40-50 h. This bioassay has been used to study herbicide-pathogen interactions, and may be extended to determine the bioherbicidal efficacy of different pathogen isolates, pathovars or spore formulations. This technique is more rapid, uses a lower inoculum volume, requires less space and is performed under more controlled conditions than conventional greenhouse bioassay methods. The data obtained are more quantitative than those obtained from bioassays relying on visual rating systems.