Australasian Plant Pathology

Spot blotch, caused by Cochliobolus sativus, is a common foliar disease of barley that is controlled primarily through the development of resistant cultivars. An assay involving the inoculation of a detached first leaf (DFL) maintained in vitro was used to determine the reaction of five barley genotypes to the disease. The estimation of the infected leaf area enabled discrimination among barley genotypes differing in their susceptibility to the pathogen. Significant correlations were found (P=0.001) between in vitro DFL values in both seedling (r=0.89) and adult (r=0.95) plants. The established assay using in vitro DFL enables a fast assessment of the susceptibility of barley to spot blotch and should be useful for many types of studies on this disease.

Pathogenicity analysis of Australian isolates of Erysiphe graminis f.sp. hordei on a set of barley lines, near-isogenic for powdery mildew resistance, and RFLP analyses of the DNA from these isolates showed only minor differences between them. The study also revealed that many ‘unnecessary genes’ for virulence have been retained. The isolates of E. graminis contained a significant quantity of dsRNA fragments, with some fragments encapsidated as virus-like particles, and a DNA plasmid homologous to a plasmid isolated previously from a European isolate. The strong homology with European genomic and plasmid DNAs, and the similarity of dsRNA, suggest that Australian isolates of E. graminis are of European origin. However, since introduction, evolution of the pathogen has diverged from that of European isolates, due to geographical isolation and lack of selection pressure from barley cultivars resistant to powdery mildew.

The disease cycle of the chili (Capsicum annuum) anthracnose fungus Colletotrichum truncatum (formerly C. capsici) was elucidated from a study of infection and colonization of seed, leaves and fruit. Microscopic observations of detached leaves and fruit inoculated with a virulent pathotype (F83B), revealed direct cuticle penetration and, intramural, endophytic and necrotrophic phases of colonization. Seedling and fruit ripening stages were very susceptible to infection with the pathogen causing pre- and post-emergence damage and postharvest fruit rot. Furthermore, a quiescent stage, following leaf infection during the vegetative phase of plant growth served as a potential primary inoculum source for fruit infection. Leaf epidermal cells of the resistant C. chinense PBC932 expressed a strong hypersensitive response 48 h after infection (HAI) to both highly virulent (F83B) and less virulent (BRIP 26,974) pathotypes. Infected cells had thickened cell walls, cytoplasm aggregation, and high levels of reactive oxygen species produced 12 HAI. In contrast, the infected epidermal cells of the susceptible C. annuum cultivar Bangchang showed necrosis and rapid cell death after infection by either pathotype. Knowledge of the disease cycle of C. truncatum will be helpful in understanding the behaviour of the pathogen in chili fields which will lead to more efficient application of control measures.

In May 1993, symptoms of asiatic citrus canker were found on West Indian lime, lemon and grapefruit trees in a mixed citrus orchard at Lambell’s Lagoon, near Darwin, Northern Territory, close to the site of a previous (1991) canker outbreak. Symptoms occurred on the spring growth flush at the start of the wet season (October 1992). The time lag between the appearance of symptoms and collection of material made it difficult to isolate the causal agent directly from the lesions, largely due to the overgrowth of secondary organisms. To overcome this difficulty we used a method for detecting Xanthomonas campestris pv. citri based on the polymerase chain reaction. A primer pair, known to amplify only DNA from group A of X. c. citri, directed the amplification of a DNA fragment of the expected size (222 base pairs, bp) from crude exudates prepared from leaf or fruit lesions, from mixed cultures, from inoculated citrus leaves and from positive control DNAs prepared from reference cultures of X. c. citri. A second primer pair and a duplex PCR were then tested, these also generated products of the expected sizes, and hence a presumptive diagnosis of asiatic citrus canker was made. We believe that this is the first use of PCR technology to diagnose a field outbreak of citrus canker. The total time from specimen preparation to detection of PCR products was less than 7 h. Colonies resembling X. citri were eventually recovered from only 2 of 11 symptomatic samples. A sensitive genomic fingerprinting technique provided strong evidence that the 1991 outbreak was the source of the current infestation.

Yellow mosaic disease of jute (Corchorus capsularis) was found to be associated with a whitefly-vectored begomovirus. Sequencing of part of the begomovirus DNA-A indicated the presence of a New World begomovirus which has been detected for the first time in India.

Downy mildew pathogen of pearl millet in India is associated with the spread of the highly virulent Sclerospora graminicola pathotype-1. Twenty-seven S. graminicola isolates were screened using 20 inter simple sequence repeats (ISSR). Dinucleotide repeat primer [17898A-(CA)6 AC] amplified a ∼600 bp fragment specific to five isolates of pathotype-1 (Sg 048, Sg 153, Sg 212, DM-11 and DM-90). The ISSR fragment linked with pathotype-1 was cloned successfully and sequenced. To convert ISSR fragments into pathotype-specific sequence characterised amplified region (SCAR) markers, PCR primers were designed using a sequence of the cloned DNA fragment. PCR amplification using SCAR primer pair (UOM3-Sg-Path1-F/R) amplified a single 284 bp band only in isolates of S. graminicola pathotype-1. This SCARprimer pair did not amplify the 284 bp product from the other five S. graminicola pathotypes or a negative control, which demonstrates primer specificity for pathotype-1. The SCAR primer pair (UOM3-Sg-Path1-F/R) obtained in this study will provide a valuable tool for rapid identification and specific detection of S. graminicola pathotype-1.

During summer and early autumn in 1989, barley yellow dwarf viruses (BYDV) were detected by ELISA in grasses collected at 72 sites in the south-west of Western Australia. Most of the grasses sampled survived the dry conditions in roadside ditches or at the edges of creeks. BYDV was found at ten or more sites in each of five perennial species, Cynodon dactylon, Ehrharta calycina, Eragrostis curvula, Paspalurn dilatatum and Pennisetum clandestinum, and in one summer annual, Digitaria sanguinalis. PAV and RPV were detected at 71% and 86% of infected sites, respectively. During the late winter and early spring in 1989, BYDV was detected in 52% of cereal crops (oats, barley and wheat) sampled in the south coastal region of Western Australia, with >5% infection being recorded in 10% of crops. Highest levels of infection within crops (up to 26%) were found in the Albany and Jerramungup districts. By contrast, the virus was found in only 29% of cereal crops sampled from the western parts of the southern wheat belt where only one crop had >2% infection. When some of the BYDV-infected cereal samples were retested, PAV and RPV were detected in 65% and 39010, respectively.

Two foliar Phytophthora species are impacting New Zealand’s pine forests. Both Phytophthora pluvialis and Phytophthora kernoviae cause similar symptoms on pine needles, however P. pluvialis is a recent arrival to New Zealand and is the primary cause of red needle cast disease in Pinus radiata. The objective of this study was to determine if Phytophthora species could be detected in archived foliage collections from plantation forests in New Zealand, to ascertain how long they have been associated with foliar disorders. Foliage samples were selected from dried collections of P. radiata and Pseudotsuga menziesii made between 1963 and 2004. From these, DNA extractions and polymerase chain reactions (PCR) were performed for both host and pathogen detection. Phytophthora species were detected in eight (of 44) pine mycological herbarium samples; of these, three contained P. kernoviae. The positive P. kernoviae samples were collected in 1999 from Northland, and in 2000 and 2002 from Gisborne. Phytophthora species were detected in an additional six archived foliage samples, collected for nutrient analysis: P. kernoviae in a 1986 sample from Riverhead, Auckland, a putative Phytophthora cactorum in a 1972 sample from Glenbervie, Northland, and other Phytophthora spp. in samples collected in 1972, 1986, 1994 and 1996. Phytophthora pluvialis was not detected in any of the samples tested. The results of this study indicate that some Phytophthora species have been present in pine foliage since at least the 1970s. The presence of P. kernoviae in pine foliage since 1986 suggests that P. kernoviae may be associated with physiological needle blight, a disorder of P. radiata with a previously unknown cause.

Tilletia indica, the cause of Karnal bunt of wheat, replaces part of the seed with a black powdery mass containing millions of spores and produces a strong unpleasant odour like rotten fish. The fishy smell has serious consequences for the marketability of wheat. The fungus is thus subjected to very strict quarantine regulations in countries not known to have the pathogen. The pathogen is spread through natural or human dissemination of spores. Research had shown that a detectable level of the disease would indicate the pre-existence of the pathogen for several years. Once present, this pathogen is extremely difficult to eradicate. Early detection at the incursion stage is thus very important to prevent disease establishment and the spread of the pathogen in a new area. Early detection in the field is unlikely. The key to its detection is thus the deployment of strategic surveillance and quarantine regulations in the wheat supply chain with the use of very sensitive and accurate diagnostic tools to detect and identify a small number of spores. This paper reviews the development of diagnostic assays of increasing sensitivity and specificity for the identification and differentiation of T. indica and other Tilletia spp. contaminants commonly found in wheat grains.