Stunted cotton (Gossypium hirsutum L.) fully recovers biomass and yield of seed cotton after delayed root inoculation with spores of an arbuscular mycorrhizal fungus (Glomus mosseae)

In the northern grain and cotton region of Australia, poor crop growth after long periods of fallow, called 'long-fallow' disorder, is caused by a decline of natural arbuscular-mycorrhizal fungi (AMF). When cotton was grown in large pots containing 22 kg of Vertisol from a field recently harvested from cotton in Central Queensland, plants in pasteurised soil were extremely stunted compared with plants in unpasteurised soil. We tested the hypothesis that this extreme stunting was caused by the absence of AMF and examined whether such stunted plants could recover from subsequent treatment with AMF spores and/or P fertiliser. At 42 days after sowing, the healthy cotton growing in unpasteurised soil had 48% of its root-length colonised with AMF, whereas the stunted cotton had none. After inoculation with AMF spores (6 spores/g soil of Glomus mosseae) and/or application of P fertiliser (50 mg P/kg soil) at 45 days after sowing, the stunted plants commenced to improve about 25 days after treatment, and continued until their total dry matter and seed cotton production equalled that of plants growing in unpasteurised soil with natural AMF. In contrast, non-mycorrhizal cotton grown without P fertiliser remained stunted throughout and produced no bolls and only 1% of the biomass of mycorrhizal cotton. Even with the addition of P fertiliser, non-mycorrhizal cotton produced only 64% of the biomass and 58% of the seed cotton (lint + seed) of mycorrhizal cotton plants. These results show that cotton is highly dependent on AMF for P nutrition and growth in Vertisol (even with high rates of P fertiliser), but can recover from complete lack of AMF and consequent stunting during at least the first 45 days of growth when treated with AMF spores and/or P fertiliser. This corroborates field observations in the northern region that cotton may recover from long-fallow disorder caused by low initial levels of AMF propagules in the soil as the AMF colonisation of its roots increases during the growing season.

Field inoculation with arbuscular-mycorrhizal fungi overcomes phosphorus and zinc deficiencies of linseed (Linum usitatissimum) in a vertisol subject to long-fallow disorder

Long-fallow disorder is expressed as exacerbated deficiencies of phosphorus (P) and/or zinc (Zn) in field crops growing after long periods of weed-free fallow. The hypothesis that arbuscular-mycorrhizal fungi (AMF) improve the P and Zn nutrition, and thereby biomass production and seed yield of linseed (Linum usitatissimum) was tested in a field experiment. A factorial combination of treatments consisting of +/- fumigation, +/- AMF inoculation with Glomus spp., +/- P and +/- Zn fertilisers was used on a long-fallowed vertisol. The use of such methods allowed an absolute comparison of plants growing with and without AMF in the field for the first time in a soil disposed to long-fallow disorder. Plant biomass, height, P and Zn concentrations and contents, boll number and final seed yield were (a) least in fumigated soil with negligible AMF colonisation of the roots, (b) low initially in long-fallow soil but increased with time as AMF colonisation of the roots developed, and (c) greatest in soil inoculated with AMF cultures. The results showed for the first time in the field that inflows of both P and Zn into linseed roots were highly dependent on %AMF-colonisation (R-2 = 0.95 for P and 0.85 for Zn, P < 0.001) in a soil disposed to long-fallow disorder. Relative field mycorrhizal dependencies without and with P+Zn fertiliser were 85 % and 86 % for biomass and 68 % and 52 % for seed yield respectively. This research showed in the field that AMF greatly improved the P and Zn nutrition, biomass production and seed yield of linseed growing in a soil disposed to long-fallow disorder. The level of mycorrhizal colonisation of plants suffering from long-fallow disorder can increase during the growing season resulting in improved plant growth and residual AMF inoculum in the soil, and thus it is important for growers to recognise the cause and not terminate a poor crop prematurely in order to sow another. Other positive management options to reduce long fallows and foster AMF include adoption of conservation tillage and opportunity cropping.

Effects of silicon treatment and inoculation with Fusarium oxysporum f. sp. vasinfectum on cellular defences in root tissues of two cotton cultivars

BACKGROUND AND AIMS: Silicon has been shown to enhance the resistance of plants to fungal and bacterial pathogens. Here, the effect of potassium silicate was assessed on two cotton (Gossypium hirsutum) cultivars subsequently inoculated with Fusarium oxysporum f. sp. vasinfectum (Fov). Sicot 189 is moderately resistant whilst Sicot F-1 is the second most resistant commercial cultivar presently available in Australia. METHODS: Transmission and light microscopy were used to compare cellular modifications in root cells after these different treatments. The accumulation of phenolic compounds and lignin was measured. KEY RESULTS: Cellular alterations including the deposition of electron-dense material, degradation of fungal hyphae and occlusion of endodermal cells were more rapidly induced and more intense in endodermal and vascular regions of Sicot F-1 plants supplied with potassium silicate followed by inoculation with Fov than in similarly treated Sicot 189 plants or in silicate-treated plants of either cultivar not inoculated with Fov. Significantly more phenolic compounds were present at 7 d post-infection (dpi) in root extracts of Sicot F-1 plants treated with potassium silicate followed by inoculation with Fov compared with plants from all other treatments. The lignin concentration at 3 dpi in root material from Sicot F-1 treated with potassium silicate and inoculated with Fov was significantly higher than that from water-treated and inoculated plants. CONCLUSIONS: This study demonstrates that silicon treatment can affect cellular defence responses in cotton roots subsequently inoculated with Fov, particularly in Sicot F-1, a cultivar with greater inherent resistance to this pathogen. This suggests that silicon may interact with or initiate defence pathways faster in this cultivar than in the less resistant cultivar.

Rhizoctonia crown and root rot of the pasture legume, sulla (Hedysarum coronarium)

Rhizoctonia solani AG-2-2 was isolated from wilting and dying plants of sulla (Hedysarum coronarium), which is currently being assessed in eastern and southern Australia for its potential as a pasture and forage legume. Infected plants in the field had extensive rotting of the taproot, lateral roots and crown. Koch's postulates were fulfilled using three inoculation methods. The disease may pose a considerable threat to the potential use of H. coronarium in the dryland, grazing farming systems of Australia, with resistance offering the most viable option for minimising its impact.

Selection and characterisation of two attenuated vaccine lines of Eimeria tenella in Australia

Objective To attenuate two strains of Eimeria tenella by selecting for precocious development and evaluate the strains in characterisation trials and by field evaluation, to choose one precocious line for incorporation into an Australian live coccidiosis vaccine for poultry. Design Two strains from non-commercial flocks were passaged through chickens while selecting for precocious development. Each strain was characterised for drug sensitivity, pathogenicity, protection against homologous and heterologous challenge, and oocyst output in replicated experiments in which the experimental unit was a cage of three birds. Oocyst output and/or body weight gain data collected over a 10 to 12 day period following final inoculation were measured. Feed conversion ratios were also calculated where possible. Results Fifteen passages resulted in prepatent periods reduced by 24 h for the Redlands strain (from 144 h to 120 h)and 23 h for the Darryl strain (from 139 h to 116 h). Characterisation trials demonstrated that each precocious line was significantly less pathogenic than its parent strain and each effectively induced immunity that protected chickens against challenge with both the parent strain and other virulent field strains. Both lines had oocyst outputs that, although significantly reduced relative to the parent strains, remained sufficiently high for commercial vaccine production, and both showed susceptibility to coccidiostats. Conclusion Two attenuated lines have been produced that exhibit the appropriate characteristics for use in an Australian live coccidiosis vaccine.

Antibody response against endogenous stages of an attenuated strain of Eimeria tenella

The application of attenuated vaccines for the prevention of chicken coccidiosis has increased exponentially in recent years. In Eimeria infections, protective immunity is thought to rely on a strong cell mediated response with antibodies supposedly playing a minor role. However, under certain conditions antibodies seem to be significant in protection. Furthermore, antibodies could be useful for monitoring natural exposure of flocks to Eimeria spp. and for monitoring the infectivity of live vaccines. Our objective was to investigate the chicken antibody response to the different parasite lifecycle stages following infection with an attenuated strain of Eimeria tenella. Western blotting analysis of parasite antigens prepared from the lining of caeca infected with the attenuated strain of E. tenella revealed two dominant antigens of 32 and 34 kDa, apparently associated with trophozoites and merozoites that were present at high concentrations between 84 and 132 h post-infection. When cryosections of caeca infected with E. tenella were probed with IgY purified from immune birds the most intense reaction was observed with the asexual stages. Western blotting analysis of proteins of purified sporozoites and third generation merozoites and absorption of stage-specific antibodies from sera suggested that a large proportion of antigens is shared by the two stages. The time-courses of the antibody response to sporozoite and merozoite antigens were similar but varied depending on the inoculation regime and the degree of oocyst recirculation.

Validation of a Competitive Enzyme-Linked Immunosorbent Assay for Detection of Babesia bigemina Antibodies in Cattle

A competitive enzyme-linked immunosorbent assay (cELISA) based on a broadly conserved, species-specific, B-cell epitope within the C terminus of Babesia bigemina rhoptry-associated protein 1a was validated for international use. Receiver operating characteristic analysis revealed 16% inhibition as the threshold for a negative result, with an associated specificity of 98.3% and sensitivity of 94.7%. Increasing the threshold to 21% increased the specificity to 100% but modestly decreased the sensitivity to 87.2%. By using 21% inhibition, the positive predictive values ranged from 90.7% (10% prevalence) to 100% (95% prevalence) and the negative predictive values ranged from 97.0% (10% prevalence) to 48.2% (95% prevalence). The assay was able to detect serum antibody as early as 7 days after intravenous inoculation. The cELISA was distributed to five different laboratories along with a reference set of 100 defined bovine serum samples, including known positive, known negative, and field samples. The pairwise concordance among the five laboratories ranged from 100% to 97%, and all kappa values were above 0.8, indicating a high degree of reliability. Overall, the cELISA appears to have the attributes necessary for international application.

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.

Concentrations of constitutive alk(en)ylresorcinols in peel of commercial mango varieties and resistance to postharvest anthracnose

Mature green mango fruits of commercially important varieties were screened to investigate the levels of constitutive antifungal compounds in peel and to assess anthracnose disease after inoculation with Colletotrichum gloeosporioides. High pressure liquid chromatography was used to quantify the levels of 5-n-heptadecenylresorcinol and 5-n-pentadecylresorcinol in the peel extracts. The fruit peel of the varieties ‘Kensington Pride’ and ‘Keitt’ were observed to have the highest levels of both 5-n-heptadecenylresorcinol (107.3-123.7 and 49.9-61.4 μg/g FW, respectively) and 5-n-pentadecylresorcinol (6.32-7.99 and 3.30-6.05 μg/g FW, respectively), and the fruit of the two varieties were found to have some resistance to postharvest anthracnose. The varieties ‘Kent’, ‘R2E2’, ‘Nam Doc Mai’, ‘Calypso’, and ‘Honey Gold’ contained much lower concentrations of resorcinols in their peel and three of these varieties were found to be more susceptible to anthracnose. Concentrations of 5-nheptadecenylresorcinol were significantly lower at the ‘sprung’ and ‘eating ripe’ stages of ripening compared to levels at harvest. Concentrations of 5-n-pentadecylresorcinol did not differ significantly across the three stages of ripening. The levels of these two resorcinols were found to be strongly inter-correlated (P < 0.001, r2 = 0.71), with concentrations of 5-nheptadecenylresorcinol being an average 18 times higher than those of 5-npentadecylresorcinol. At the ‘eating ripe’ stage, significant relationships were observed between the concentrations of each type of alk(en)ylresorcinol and anthracnose lesion areas following postharvest inoculation, P<0.001, r2= 0.69 for 5-n pentadecylresorcinol, and P<0.001, r2= 0.44 for 5-n-heptadecenylresorcinol.

Infection, colonisation and sporulation by Pseudocercospora macadamiae on macadamia fruit

The structures and manner with which Pseudocercospora macadamiae penetrates, colonises and proliferates from the pericarp of macadamia fruit was studied using scanning electron microscopy and fluorescence light microscopy. Germ tubes arising from conidia penetrated open stomata within 20 h of inoculation, without observation of specialised infection structures such as appressoria. Colonisation of the pericarp was intercellular, without observation of specialised intracellular infection structures such as haustoria, and was complete from the epidermis to the mesocarp. The fungus proliferated at the epidermis by the formation of conidiophores and conidia on substomatal and protuberant subepidermal stromata. These structures were not observed on the mesocarp surface. The onset of visual husk spot symptoms coincided with an increase in pathogen biomass on the pericarp surface. The progression of symptoms from tan-coloured spots to larger red-brown lesions coincided with the production of conidiophores from substomatal and protuberant subepidermal stromata. The darker the colour of the husk spot lesion, the more frequently protuberant subepidermal stromata were observed. These findings are discussed in the context of observation of other cercosporoid fungi.

Use of optical density as a measure of Claviceps africana conidial suspension concentration

Sorghum ergot, caused by Claviceps africana, has remained a major disease problem in Australia since it was first recorded in 1996, and is the focus of a range of biological and integrated management research. Artificial inoculation using conidial suspensions is an important tool in this research. Ergot infection is greatly influenced by environmental factors, so it is important to reduce controllable sources of variation such as inoculum concentration. The use of optical density was tested as a method of quantifying conidial suspensions of C. africana, as an alternative to haemocytometer counts. This method was found to be accurate and time efficient, with possible applications in other disease systems.

Cucumber mosaic virus infection of kava (Piper methysticum) and implications for cultural control of kava dieback disease

Cucumber mosaic virus (CMV) was found by reverse transcription polymerase chain reaction (RT-PCR) to be not fully systemic in naturally infected kava (Piper methysticum) plants in Fiji. Twenty-six of 48 samples (54%) from various tissues of three recently infected plants were CMV-positive compared with 7/51 samples (14%) from three long-term infections (plants affected by dieback for more than 1 year). The virus was also found to have a limited ability to move into newly formed stems. CMV was detected in only 2/23 samples taken from re-growth stems arising from known CMV infected/dieback affected plants. Mechanical inoculation experiments conducted in Fiji indicate that the known kava intercrop plants banana (Musa spp.), pineapple (Ananas comosus), peanut (Arachis hypogaea) and the common weed Mikania micrantha are potential hosts for a dieback-causing strain of CMV It was not possible to transmit the virus mechanically to the common kava intercrop plants taro (Colocasia esculenta), Xanthosoma sp., sweet potato (Ipomoea batatas), yam (Dioscorea alata), papaya (Carica papaya) or the weed Momordica charantia. Implications of the results of this research on a possible integrated disease management strategy are discussed.

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.

Australian Bat Lyssavirus

In Chapter 1, the literature relating to rabies virus and the rabies like lyssaviruses is reviewed. In Chapter 2, data are presented from 1170 diagnostic submissions for ABLV testing by fluorescent antibody test (Centocor FAT). All 27 non-bat submissions were ABLV-negative. Of 1143 bat accessions 74 (16%) were ABLV-positive, including 69 of 974 (7.1%) flying foxes (Pteropus spp.), 5 of 7 (71.4%) Saccolaimus flaviventris (Yellow-bellied sheathtail bats), none of 151 other microchiropteran bats, and none of 11 unidentified bats. Statistical analysis of data from 868 wild Black, Grey-headed, Little Red and Spectacled flying foxes (Pteropus alecto, P. poliocephalus, P. scapulatus, and P. conspicillatus) indicated that three factors; species, health status and age were associated with significant (p< 0.001) differences in the proportion of ABLV-positive bats. Other factors including sex, whether the bat bit a person or animal, region, year, and season submitted, were not associated with ABLV. Case data for 74 ABLV-positive bats, including the circumstances in which they were found and clinical signs, is presented. In Chapter 3, the aetiological diagnosis was investigated for 100 consecutive flying fox submissions with neurological signs. ABLV (32%), spinal and head injuries (29%), and neuro-angiostrongylosis (18%) accounted for most neurological syndromes in flying foxes. No evidence of lead poisoning was found in unwell (n=16) or healthy flying foxes (n=50). No diagnosis was reached for 16 cases, all of which were negative for ABLV by TaqMan PCR. The molecular diversity of ABLV was examined in Chapter 4 by sequencing 36 bases of the leader sequence, the entire N gene, and start of the P gene of 28 isolates from pteropid bats and 3 isolates from Yellow-bellied sheathtail (YBST) bats. Phylogenetic analysis indicated all ABLV isolates clustered together as a discrete group within the Lyssavirus genera closely related to rabies virus and European bat lyssavirus-2 isolates. The ABLV lineage consisted of two variants; one (ybst-ABLV) consisted of isolates only from YBST bats, the other (pteropid-ABLV) was common to Black, Grey-headed and Little Red flying foxes. No associations were found between the sequences and either the geographical location or year found, or individual flying fox species. In Chapter 5, 15 inocula prepared from the brains or salivary glands of naturally-infected bats were evaluated by intracerebral (IC) and footpad (FP) inoculation of Quackenbush mice in order to select and characterize a highly virulent inoculum for further use in bats (Inoculum 5). In Chapter 6, nine Grey-headed flying foxes were inoculated with 105.2 to 105.5 MICED50 of Inoculum 5 divided into four sites, left footpad, pectoral muscle, temporal muscle and muzzle. Another bat was inoculated with half this dose divided into the footpad and pectoral muscle only. Seven of 10 bats developed clinical disease of 1 to 4 days duration between PI-days 10 and 19 and were shown to be ABL-positive by FAT, HAM immunoperoxidase staining, virus isolation in mice, and TaqMan PCR. Five of the seven bats displayed overt aggression, one died during a seizure, and one showed intractable agitation, pacing, tremors, and ataxia. Viral antigen was demonstrated throughout the central and peripheral nervous systems and in the epithelial cells of the submandibular salivary glands (n=4). All affected bats had mild to moderate non-suppurative meningoencephalitis and severe ganglioneuritis. No ABLV was detected in three bats that remained well until the end of the experiment on day 82. One survivor developed a strong but transient antibody response. In Chapter 7, the relative virulence of inocula prepared from the brains and salivary glands of experimentally infected flying foxes was evaluated in mice by IC and FP inoculation and TaqMan assay. The effects in mice were correlated to the TaqMan CT value and indicated a crude association between virulence and CT value that has potential application in the selection of inocula. In Chapter 8, 36 Black and Grey-headed flying foxes were vaccinated with one (day 0) or two (+ day 28) doses of Nobivac rabies vaccine and co-vaccinated with keyhole limpet haemocyanin (KLH). All bats responded to the Nobivac vaccine with a rabies-RFFIT titer > 0.5 IU/mL that is nominally indicative of protective immunity. Plasma from bats with rabies titres >2 IU/mL had cross-neutralising ABLV titres >1:154. A specifically developed ELISA detected a strong but transient response to KLH.

Transmission of Japanese Encephalitis Virus from the Black Flying Fox, Pteropus alecto, to Culex annulirostris Mosquitoes, Despite the Absence of Detectable Viremia.

To determine the potential role of flying foxes in transmission cycles of Japanese encephalitis virus (JEV) in Australia, we exposed Pteropus alecto (Megachiroptera: Pteropididae) to JEV via infected Culex annulirostris mosquitoes or inoculation. No flying foxes developed symptoms consistent with JEV infection. Anti-JEV IgG antibodies developed in 6/10 flying foxes exposed to infected Cx. annulirostris and in 5/5 inoculated flying foxes. Low-level viremia was detected by real-time reverse transcriptase polymerase chain reaction in 1/5 inoculated flying foxes and this animal was able to infect recipient mosquitoes. Although viremia was not detected in any of the 10 flying foxes that were exposed to JEV by mosquito bite, two animals infected recipient mosquitoes. Likewise, an inoculated flying fox without detectable viremia infected recipient mosquitoes. Although infection rates in recipient mosquitoes were low, the high population densities in roosting camps, coupled with migratory behavior indicate that flying foxes could play a role in the dispersal of JEV.