Development of a small-plant bioassay to assess banana grown from tissue culture for consistent infection by Fusarium oxysporum f. sp. cubense

Two reliable small-plant bioassays were developed using tissue-cultured banana, resulting in consistent symptom expression and infection by Fusarium oxysporum f. sp. cubense (Foc). One bioassay was based on providing a constant watertable within a closed pot and the second used free-draining pots. Culture medium for spore generation influenced infectivity of Foc. Inoculation of potted banana by drenching potting mix with a conidial suspension, consisting mostly of microconidia, few macroconidia and no chlamydospores, generated from one-quarter-strength potato dextrose agar + streptomycin sulfate, resulted in inconsistent infection. When a conidial suspension that consisted of all three spore types, microconidia, macroconidia and chlamydospores, prepared from spores generated on carnation leaf agar was used, all plants became infected, indicating that the spore type present in conidial suspensions may contribute to inconsistency of infection. Inconsistency of infection was not due to loss of virulence of the pathogen in culture. Millet grain precolonised by Foc as a source of inoculum resulted in consistent infection between replicate plants. Sorghum was not a suitable grain for preparation of inoculum as it was observed to discolour roots and has the potential to stunt root growth, possibly due to the release of phytotoxins. For the modified closed-pot system, a pasteurised potting mix consisting of equal parts of bedding sand, perlite and vermiculite plus 1 g/L Triabon slow release fertiliser was suitable for plant growth and promoted capillary movement of water through the potting mix profile. A suitable potting mix for the free-draining pot system was also developed.

Infection by Alternaria alternata causes discolouration of Backhousia myrtifolia foliage and flowers.

The pathogenicity of three isolates of Alternaria alternata from Backhousia myrtifolia leaves was characterised and compared. Isolate BRIP 52222 was virulent compared to isolates BRIP 52223 and BRIP 52221. A comparison of inoculation methods showed that abrasion was more effective at establishing an infection than puncture wounding. Koch's postulates were assessed to confirm the pathogenicity of A. alternata on B. myrtifolia foliage and floral tissues using a conidial suspension of the most virulent isolate. Sporulation was triggered by incubating A. alternata (BRIP 52222) at 28 degrees C for 10 d under alternating 12 h black-light/12 h dark conditions on half-strength potato dextrose agar (PDA). In contrast, incubation of A. alternata under continuous black-light on either half- or full-strength PDA did not yield conidia. Host symptoms caused by inoculation with the pathogen included a brown-black discolouration of both foliage and floral tissues. Microscopic examination of cellular structures suggested that perturbation of oil glands may contribute to the tissue discolouration in B. myrtifolia caused by A. alternata infection. Oil gland structures can be disrupted during an active A. alternata infection, causing the leakage of essential oil followed by discolouration.

Characterisation of accessions and species of Macadamia to stem infection by Phytophthora cinnamomi

Phytophthora cinnamomi is a major pathogen in most macadamia plantations worldwide. Due to stem lesions, stem cankers and leaf defoliation it results in loss of productivity and tree death. In this study we examined accessions of the four Macadamia species and their hybrids, produced via rooted stem cuttings or germinated seeds, for susceptibility to stem canker and necrotic lesion caused by P. cinnamomi. Plants were wound-inoculated with agar containing P. cinnamomi. The symptoms produced in inoculated plants were used to characterize host susceptibility variation within and among the population. Lesion lengths and severity of stem canker were recorded. The four species and hybrids differed significantly in stem canker severity (P < 0.001) and lesion length (P = 0.04). M. integrifolia and M. tetraphylla hybrids were the most susceptible. M. integrifolia had the greatest stem canker severity and the most extensive lesions above and below the site of inoculation. Restricted lesion sizes were observed in M. ternifolia and M. jansenii. The effects of basal stem diameter and the method of propagation either from cuttings or seed were not significant. The genetic variation in the reactions of macadamia accessions to stem infection by P. cinnamomi is discussed.

In vitro infection of sheep lice (Bovicola ovis Schrank) by Steinernematid and Heterorhabditid nematodes.

Control of sheep lice with conventional pesticides can be compromised by difficulty in contacting lice in the dense water repellent fleeces of sheep, particularly when sheep have not been recently shorn. Entomopathogenic nematodes (ENs) are motile and are able to actively seek out insect hosts. They have particular advantages for the control of pests in cryptic habitats, such as the fleeces of sheep and avoid many of the problems frequently associated with chemical controls. This study investigated whether ENs were able infect and kill Bovicola ovis and compared the effectiveness of different species at different temperatures and when applied to wool. Four species of nematodes, Steinernema carpocapsae, Steinernema riobrave, Steinernema feltiae and Heterorhabditis bacteriophora were tested. All were shown to infect and kill lice in Petri dish assays at 30C. At 35C, the percent infection for S. carpocapsae and S. riobrave was significantly higher than for the other two species and percent infection by S. feltiae was significantly greater than for H. bacteriophora (P<0.05). At 37C the percent mortality induced by S. riobrave was significantly greater than for S. carpocapsae (P<0.05). All species were able to locate and infect lice in wool when formulated in water with 8% Tween 80. In wool assays the percent lice infected with nematodes was significantly greater for S. riobrave than H.bacteriophora at 25C, but there were no other differences between species (P=0.05). S. carpocapsae, S. riobrave and S. feltiae caused significantly higher lice mortality than H. bacteriophora at both 25 and 35C in wool assays, but mortality induced by the three steinernematid species did not differ significantly (P>0.05). It is concluded that of the ENs studied S. riobrave is likely to be most effective against B. ovis when applied to live sheep because of its greater tolerance to high temperatures and 'cruiser' foraging strategy .

Rejection by pigs of mouldy grain containing deoxynivalenol

Weaner pigs on a farm near Beaudesert in south eastern Queensland refused to eat feed comprised largely of wheat and barley. Older pigs consumed small amounts and some prepubertal gilts subsequently displayed enlarged and reddened vulvas. Wheat, barley and triticale were grown on the farm during 1983, which was unusually and persistently wet. The wheat and triticale were harvested and stored for about 3 weeks with moisture contents above 14% before being fed. Samples of the wheat and triticale contained pale pink grains, which can indicate infection by the fungus Fusariurn grarninearurn Schw. On analysis 2 mycotoxins known to be produced by F. graminearurn were detected, deoxynivalenol (vomitoxin) which causes feed refusal and vomiting, and zearalenone which causes oestrogenic effects. Concentrations of deoxynivalenol in the wheat, triticale and barley were 34, 10, and <0.1 mg/kg respectively. Concentrations of zearalenone were 6.2, 2.8 and 0.1 mg/kg respectively. Subsequently, F. grarninearurn was isolated from grains and crop residues. Although the wet weather contributed to F. grarninearurn infection of the crops before harvest, most of the toxins probably developed during storage.

Tan spot: A new disease of pyrethrum caused by Microsphaeropsis tanaceti sp. nov

The isolation frequency of Microsphaeropsis sp. in spring in association with necrotic lesions on leaves in Tasmanian pyrethrum (Tanacetum cinerariifolium) fields has increased substantially since first identification in 2001. Examination of morphological features and sequencing of the internal transcribed spacer region (ITS) resulted in the identification of a new species, herein described as Microsphaeropsis tanaceti sp. nov. The pathogenicity of three M. tanaceti isolates to two pyrethrum cultivars was confirmed by inoculating glasshouse-grown plants in three experiments. No significant differences in the susceptibility of the two cultivars to infection by M. tanaceti were found. Symptoms were tan-coloured spots which coalesced around the margins of the leaves. Therefore, the name 'tan spot' is proposed for this new disease of pyrethrum. The sensitivity of seven M. tanaceti isolates to difenoconazole and azoxystrobin, commonly used fungicides for the management of foliar diseases in spring, was assessed under in vitro conditions. Sensitivity testing for difenoconazole was conducted using a mycelial growth assay on potato dextrose agar, whilst testing for sensitivity to azoxystrobin used a conidial germination assay on water agar. Microsphaeropsis tanaceti was found to be more sensitive to azoxystrobin than difenoconazole, with complete inhibition of conidial germination at concentrations above 0.625 µg a.i. mL-1. By comparison, concentrations of 50 µg a.i. difenoconazole mL-1 or greater were required for significant inhibition of mycelial growth. It therefore appears likely that there is currently some control of tan spot as a result of the use of azoxystrobin and to a lesser extent, difenoconazole, for the control of other diseases.

Kirramyces viscidus sp. nov., a new eucalypt pathogen from tropical Australia closely related to the serious leaf pathogen, Kirramyces destructans

Kirramyces destructans is a serious pathogen causing a leaf, bud and shoot blight disease of Eucalyptus plantations in the subtropics and tropics of South-East Asia. During surveillance of eucalypt taxa trials in northern Queensland, symptoms resembling those of K. destructans were observed on Eucalyptus grandis and E. grandis × E. camaldulensis. Phylogenetic and morphological studies revealed that the Kirramyces sp. associated with these symptoms represents a new taxon described here as K. viscidus sp. nov., which is closely related to K. destructans. Plantation assessments revealed that while E. grandis from the Copperload provenance, collected in northern Queensland, recovered from disease, E. grandis × E. camaldulensis hybrids from South America were highly susceptible to infection by K. viscidus and are not recommended for planting in northern Queensland. Preliminary results suggest the fungus probably originates from Australia. K. viscidus is closely related to K. destructans and causes a disease with similar symptoms, suggesting that it could seriously damage Australian eucalypt plantations, especially those planted off-site.

Molecular characterisation, pathogenesis and fungicide sensitivity of Pythium spp. from table beet (Beta vulgaris var. vulgaris) grown in the Lockyer Valley, Queensland

Table beet production in the Lockyer Valley of south-eastern Queensland is known to be adversely affected by soilborne root disease from infection by Pythium spp. However, little is known regarding the species or genotypes that are the causal agents of both pre- and post-emergence damping off. Based on RFLP analysis with HhaI, HinfI and MboI of the PCR amplified ITS region DNA from soil and diseased plant samples, the majority of 130 Pythium isolates could be grouped into three genotypes, designated LVP A, LVP B and LVP C. These groups comprised 43, 41 and 7% of all isolates, respectively. Deoxyribonucleic acid sequence analysis of the ITS region indicated that LVP A was a strain of Pythium aphanidermatum, with greater than 99% similarity to the corresponding P. aphanidermatum sequences from the publicly accessible databases. The DNA sequences from LVP B and LVP C were most closely related to P. ultimum and P. dissotocum, respectively. Lower frequencies of other distinct isolates with unique RFLP patterns were also obtained with high levels of similarity (>97%) to P. heterothallicum, P. periplocum and genotypes of P. ultimum other than LVP B. Inoculation trials of 1- and 4-week-old beet seedlings indicated that compared with isolates of the LVP B genotype, a higher frequency of LVP A isolates caused disease. Isolates with the LVP A, LVP B and LVP C genotypes were highly sensitive to the fungicide Ridomil MZ, which suppressed radial growth on V8 agar between approximately four and thirty fold at 5 μg/mL metalaxyl and 40 μg/mL mancozeb, a concentration far lower than the recommended field application rate.

Quambalaria species: increasing threat to eucalypt plantations in Australia

Spotted gum (Corymbia citriodora subsp. variegata and C. maculata) is a valuable source of commercial timber and suitable for a wide range of different soil types in eastern Australia. The main biological constraint to further expansion of spotted gum plantations is Quambalaria shoot blight caused by the fungus Quambalaria pitereka. Surveys conducted to evaluate the impact of Quambalaria shoot blight have shown that the disease is present in all spotted gum plantations and on a range of Corymbia species and hybrids in subtropical and tropical regions surveyed in eastern Australia. More recently, Q. eucalypti has also been identified from a range of Eucalyptus species in these regions. Both pathogens have also been found associated with foliage blight and die-back of amenity trees and Q. pitereka in native stands of Corymbia species, which is the probable initial infection source for plantations. Infection by Q. pitereka commonly results in the repeated destruction of the growing tips and the subsequent formation of a bushy crown or death of trees in severe cases. In comparison, Q. eucalypti causes small, limited lesions and has in some cases been associated with insect feeding. It has not been recorded as causing severe shoot and stem blight. A better understanding of factors influencing disease development and host-pathogen interactions is essential in the development of a disease management strategy for these poorly understood but important pathogens in the rapidly expanding eucalypt (Corymbia and Eucalyptus spp.) plantation industry in subtropical and tropical eastern Australia.

Significant features of the epidemiology of equine influenza in Queensland, Australia, 2007.

An outbreak of equine influenza (EI) caused by influenza A H3N8 subtype virus occurred in the Australian states of Queensland and New South Wales in August 2007. Infection in the Australian horse population was associated with the introduction of infection by horses from overseas. The first case of EI in Queensland was detected on 25 August 2007 at an equestrian sporting event. Infection subsequently spread locally and to other clusters through horse movements prior to the implementation of an official standstill. There were five main clusters of infected properties during this outbreak and several outliers, which were investigated to find the potential mechanism of disease spread. To contain the outbreak, Queensland was divided into infection status zones, with different movement controls applied to each zone. Vaccination was implemented strategically in infected areas and within horse subpopulations. Control and eventual eradication of EI from Queensland was achieved through a combination of quarantine, biosecurity measures, movement control, rapid diagnostic testing and vaccination.

Screening bacterial metabolites for inhibitory effects against Batrachochytrium dendrobatidis using a spectrophotometric assay

Certain bacteria present on frog skin can prevent infection by the pathogenic fungus Batrachochytrium dendrobatidis (Bd), conferring disease resistance. Previous studies have used agar-based in vitro challenge assays to screen bacteria for Bd-inhibitory activity and to identify candidates for bacterial supplementation trials. However, agar-based assays can be difficult to set up and to replicate reliably. To overcome these difficulties, we developed a semi-quantitative spectrophotometric challenge assay technique. Cell-free supernatants were prepared from filtered bacterial cultures and added to 96-well plates in replicated wells containing Bd zoospores suspended in tryptone-gelatin hydrolysate-lactose (TGhL) broth medium. Plates were then read daily on a spectrophotometer until positive controls reached maximum growth in order to determine growth curves for Bd. We tested the technique by screening skin bacteria from the Australian green-eyed tree frog Litoria serrata. Of bacteria tested, 31% showed some degree of Bd inhibition, while some may have promoted Bd growth, a previously unknown effect. Our cell-free supernatant challenge assay technique is an effective in vitro method for screening bacterial isolates for strong Bd-inhibitory activity. It contributes to the expanding field of bioaugmentation research, which could play a significant role in mitigating the effects of chytridiomycosis on amphibians around the world.

Bacterial wilt of ginger in Queensland: Reappraisal of a disease outbreak

In 1955 a severe wilt disease occurring on ginger in the Near North Coast district of Queensland was incorrectly attributed to infection by a Fusarium sp., and later shown to be caused by a strain of Ralstonia solanacearum, now reclassified as R. sequeirae. The disease was brought from China into Australia on latently infected rhizomes, and possibly also with associated soil. Several DNA-based diagnostic methods have shown that the pathogen causing bacterial wilt of ginger in parts of China is indistinguishable from the pathogen uniquely associated with the disease in Queensland. © 2012 Australasian Plant Pathology Society Inc.

Symptom development proceeds at different rates in susceptible and partially resistant cereal seedlings infected with Fusarium pseudograminearum

Variation in the reaction of cereal cultivars to crown rot caused by Fusarium spp., in particular Fusarium pseudograminearum, was identified over 50 yrs ago, however the parameters and pathways of infection by F. pseudograminearum remain poorly understood. Seedlings of wheat, barley and oat genotypes that differ in susceptibility to crown rot were inoculated with a mixture of F. pseudograminearum isolates. Seedlings were harvested from 7 to 42 days after inoculation and expanded plant parts were rated for severity of visible disease symptoms. Individual leaf sheaths were placed onto nutrient media and fungal colonies emerging from the leaf sheathes were counted to estimate the degree of fungal spread within the host tissue. Significant differences in both the timing and the severity of disease symptoms were observed in the leaf sheath tissues of different host genotypes. Across all genotypes and plant parts examined, the development of visible symptoms closely correlated with the spread of the fungus into that tissue. The degree of infection of the coleoptile and sub-crown internode varied between genotypes, but was unrelated to the putative resistance of the host. In contrast leaf sheath tissues of the susceptible barley cv. Tallon and bread wheat cv. Puseas scored higher disease ratings and consistently showed faster, earlier spread of the fungus into younger tissues than infections of the oat cv. Cleanleaf or the wheat lines 2-49 and CPI 133814. While initial infections usually spread upwards from near the base of the first leaf sheath, the pathogen did not appear to invade younger leaf sheaths only from the base, but rather spread laterally across from older leaf sheaths into younger, subtended leaf sheaths, particularly as disease progressed. Early in the infection of each leaf sheath, disease symptoms in the partially resistant genotypes were less severe than in susceptible genotypes, however as infected leaf sheaths aged, differences between genotypes lessened as disease symptoms approached maximum values. Hence, while visual scoring of disease symptoms on leaf sheaths is a reliable comparative measure of the degree of fungal infection, differences between genotypes in the development of disease symptoms are more reliably assessed using the most recently expanded leaf sheaths.

Markers for seedling and adult plant crown rot resistance in four partially resistant bread wheat sources

Key message: QTLidentified for seedling and adult plant crown rot resistance in four partially resistant hexaploid wheat sources. PCR-based markers identified for use in marker-assisted selection. Abstract: Crown rot, caused by Fusarium pseudograminearum, is an important disease of wheat in many wheat-growing regions globally. Complete resistance to infection by F. pseudograminearum has not been observed in a wheat host, but germplasm with partial resistance to this pathogen has been identified. The partially resistant wheat hexaploid germplasm sources 2-49, Sunco, IRN497 and CPI133817 were investigated in both seedling and adult plant field trials to identify markers associated with the resistance which could be used in marker-assisted selection programs. Thirteen different quantitative trait loci (QTL) conditioning crown rot resistance were identified in the four different sources. Some QTL were only observed in seedling trials whereas others appeared to be adult plant specific. For example while the QTL on chromosomes 1AS, 1BS, and 4BS contributed by 2-49 and on 2BS contributed by Sunco were detected in both seedling and field trials, the QTL on 1DL present in 2-49 and the QTL on 3BL in IRN497 were only detected in seedling trials. Genetic correlations between field trials of the same population were strong, as were correlations between seedling trials of the same population. Low to moderate correlations were observed between seedling and field trials. Flanking markers, most of which are less than 10 cM apart, have now been identified for each of the regions associated with crown rot resistance.

A retrospective study of Babesia macropus associated with morbidity and mortality in eastern grey kangaroos (Macropus giganteus) and agile wallabies (Macropus agilis)

This is a retrospective study of 38 cases of infection by Babesia macropus, associated with a syndrome of anaemia and debility in hand-reared or free-ranging juvenile eastern grey kangaroos (Macropus giganteus) from coastal New South Wales and south-eastern Queensland between 1995 and 2013. Infection with B. macropus is recorded for the first time in agile wallabies (Macropus agilis) from far north Queensland. Animals in which B. macropus infection was considered to be the primary cause of morbidity had marked anaemia, lethargy and neurological signs, and often died. In these cases, parasitised erythrocytes were few or undetectable in peripheral blood samples but were sequestered in large numbers within small vessels of visceral organs, particularly in the kidney and brain, associated with distinctive clusters of extraerythrocytic organisms. Initial identification of this piroplasm in peripheral blood smears and in tissue impression smears and histological sections was confirmed using transmission electron microscopy and molecular analysis. Samples of kidney, brain or blood were tested using PCR and DNA sequencing of the 18S ribosomal RNA and heat shock protein 70 gene using primers specific for piroplasms. The piroplasm detected in these samples had 100 sequence identity in the 18S rRNA region with the recently described Babesia macropus in two eastern grey kangaroos from New South Wales and Queensland, and a high degree of similarity to an unnamed Babesia sp. recently detected in three woylies (Bettongia penicillata ogilbyi) in Western Australia.