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Freckle disease of banana is caused by three closely related species of Phyllosticta, namely P. musarum, P. maculata and P. cavendishii. In this study, a high resolution melting (HRM) analysis assay was developed and its potential to identify these three fungal species is reported. The assay, which targets the ITS of the nuclear rDNA of the fungal species, generates three distinct melt profiles for the three Phyllosticta species. It is also able to distinguish a combination of up to three co-infecting species by generating a deviant melt curve. Thirty-five fungal cultures and infected herbarium leaf specimens, previously characterized using nucleotide sequencing as belonging to one of the three Phyllosticta species, were used for validation of the HRM analysis assay. The normalized curves generated differentiated all samples, with samples from each species correctly identified. The assay was further evaluated against 18 uncharacterized infected leaf specimens from various geographic locations and the results were verified by subsequent nucleotide sequencing. This HRM analysis assay allows rapid identification and differentiation of the three Phyllosticta species using a single primer pair in a one-step closed-tube system without labelled fluorescence probes. This novel assay format has potential for simultaneously identifying and differentiating other closely related species of plant pathogens, as well as the classification of infected historic specimens.

The initial infection stages of Phyllosticta maculata on banana were studied using scanning electron microscopy. Conidial germination on the banana leaf surface commenced within 3h postinoculation to produce a long and slender germ tube. The hyphae developed secondary branches and mostly grew randomly across the leaf surface. Appressoria were formed at the apex of the germ tubes within 18h postinoculation and were variable in shape. A layer of an extracellular matrix surrounded the appressoria at the pathogenhost interface. On the fruit surface, conidia germinated to produce predominantly swollen germ tubes which functioned as lateral appressoria together with some slender ones. These germ tubes were formed within 3h postinoculation. There was no stomatal penetration apparent on the leaf; instead, direct penetration through the cuticle with and without the formation of appressoria was observed. Cuticular degradation on the leaf surface was evident with a circular, darkened area around the point of penetration by hyphae or appressoria. The significant role of pycnidia and conidia in the epidemiology of the disease was further demonstrated in naturally infected leaf samples.

Acephate is poorly sorbed to soil, thus the risk of leaching to the aquatic environment is high if it is not quickly degraded. The effect of soil moisture, temperature, microbial activity and application rate on acephate degradation has been studied in three Malaysian soils to examine and identify critical variables determining its degradation and mineralization kinetics. First-order kinetics could be used to describe degradation in all cases ( r 2 > 0.91). Acephate degraded faster in air-dry ( t ½ 9–11 d) and field capacity ( t ½ 10–16 d) soils than in the wet soils ( t ½ 32–77 d). The activation energy of degradation was in the range 17–28 kJ mol −1 and significantly higher for the soil with higher pH and lower clay and iron oxide contents. Soil sterilization caused a 3- to 10-fold decrease in degradation rates compared to non-sterile soils ( t ½ 53–116 d) demonstrating that acephate degradation is mainly governed by microbial processes. At 5-fold increase in application rates (25 μg g −1 ), half-life increased slightly ( t ½ 13–19 d) or was unaffected. Half-life from acephate mineralization was similar to those from degradation but much longer at the 5-fold increase in acephate application rates ( t ½ 41–96 d) demonstrating that degradation of metabolites is rate limiting. Thus, application of acephate should be restricted or avoided during wet seasons with heavy rainfall and flooded soil as in paddy cultivation. Sandy soils with low microbial activity are more prone to acephate leaching than clayey soils rich in humic matter.

The phosphate analogue phosphite is widely used to control diseases of plants caused by oomycete pathogens such as those within the genus Phytophthora. Phosphite inhibits zoospore production and growth of P. cinnamomi. However, very little is known about the underlying mechanism of action. In the present study, we grew P. cinnamomi in Ribero’s liquid medium with 0.1 mM phosphate, with and without 5 μg phosphite/mL, and used differential display reverse transcriptase-PCR (DDRT-PCR) to identify P. cinnamomi genes that are transcriptionally repressed or induced by phosphite. By using this technique, four differentially expressed bands were identified. However, quantitative measurement of the amount of mRNA transcript by RT-PCR revealed that only one gene was actually phosphite inducible. On the basis of the homology of the deduced amino acid sequence, this gene encodes a proteophosphoglycan. The remaining three bands did not show differential expression.