Elsinoe perseae, the causal agent of avocado scab disease, is a high-priority biosecurity threat to the Australian avocado industry. Symptom-based diagnosis is unreliable, and morphological identification is hindered by the slow growth of Elsinoe species on artificial media and the absence of a sexual reproductive stage in the field and in axenic culture. Consequently, a rapid and reliable molecular diagnostic tool capable of detecting the pathogen directly from infected fruit or leaf tissue is essential. In this study, we developed a quantitative PCR (qPCR) assay targeting two genomic regions: the internal transcribed spacer 1 (ITS1) region of the rDNA and the RNA polymerase II subunit B (rpb2) gene. The assay can be applied in either singleplex or duplex formats. Both the ITS- and rpb2-targeted assays consistently detected E. perseae DNA from pure cultures. However, detection in infected avocado leaf and fruit tissues was primarily driven by the ITS assay, whereas the rpb2 assay exhibited reduced sensitivity, likely reflecting differences in copy number between multicopy rDNA regions and the single-copy rpb2 gene. No cross-reactivity was observed against other Elsinoe species or common fungal pathogens of avocado. High analytical sensitivity was achieved, with reliable detection of both targets at concentrations of 10³ copies/µL of synthetic gBlocks dsDNA and 0.1 ng/µL of genomic DNA, using a conservative threshold of Ct ≤ 30. Assay performance was unaffected by minor variations in qPCR reagents and instrument platforms. This assay provides a rapid, sensitive, and highly specific diagnostic tool for biosecurity applications, supporting accurate detection, surveillance, and early response to potential incursions of E. perseae.

An easy, rapid and inexpensive method of preparing RNA template for a reverse transcription qPCR assay for avocado sunblotch viroid (ASBVd) is described. This method depends on the principle of reversible binding of viroid RNA to filter paper under different concentrations of monovalent cation. Lysis buffers containing either sodium chloride or lithium chloride were compared, and 1.5 M lithium chloride was shown to be optimal for the adsorption of the viroid RNA to the filter paper. The extraction method was validated using field samples and equivalent yields of viroid RNA were obtained using this method and either a commercial RNA extraction kit or a dsRNA chromatography method. The filter paper method of RNA extraction is ideally suited for the large-scale surveillance for ASBVd.

Black root rot of avocado is a severe disease of nursery trees and young orchard transplants, causing tree death within a year after planting. In Australia, key pathogens include species complexes Calonectria ilicicola and Dactylonectria macrodidyma; however, several other Dactylonectria species also cause the disease. Rapid detection of these pathogens in planta is important to speed up implementation of disease management and reduce loss. The purpose of this study was to develop three loop-mediated isothermal amplification (LAMP) diagnostic assays to rapidly identify species within the C. ilicicola and D. macrodidyma complexes and species in the Dactylonectria genus in avocado roots. Primers were designed from β-tubulin sequence data of C. ilicicola and from histone H3 of D. macrodidyma and the Dactylonectria genus. The LAMP primers were tested for specificity and sensitivity with 82 fungal isolates, which included the target species complexes C. ilicicola and D. macrodidyma; species within the target Dactylonectria genus viz. D. macrodidyma, D. anthuriicola, D. novozelandica, D. pauciseptata, and D. vitis; and isolates of nontarget species, including Calonectria sp., Cylindrocladiella sp., Gliocladiopsis forsbergii, G. peggii, G. whileyi, Ilyonectria sp., Mariannaea sp., Fusarium sp., and Phytophthora cinnamomi. The species-specific LAMP assays were sensitive and specific at DNA concentrations of 1 pg/µl for C. ilicicola and 0.01 ng/µl for D. macrodidyma, whereas the Dactylonectria genus-wide assay was sensitive to 0.1 ng/µl. Detection of C. ilicicola occurred within 10 to 15 or 15 to 30 min when the template was pure DNA or crude extracts obtained from suspending fungal cultures in sterile water, respectively. Detection of D. macrodidyma was between 12 to 29 min with pure DNA and 16 to 30 min with crude extracts. Dactylonectria spp. were detected within 6 to 25 min with pure DNA and 7 to 23 min with crude extracts. The specificity of the assays was found to be dependent on time and isothermal amplification temperature, with optimal specificity occurring in reactions of <30 min and at temperatures of 67°C for C. ilicicola and D. macrodidyma assays and 69°C for Dactylonectria genus-wide assays. The assays were modified to accommodate a DNA extraction step and use of avocado roots as DNA templates. Detection in avocado roots ranged between 12 to 25 min for C. ilicicola, 12 to 26 min for D. macrodidyma, and 14 to 30 min for species in the Dactylonectria genus. The LAMP assays are applicable across multiple agricultural industries, because C. ilicicola, D. macrodidyma, and Dactylonectria spp. are also important pathogens of various crops and ornamental plants.

The Botryosphaeriales, and in particular the Botryosphaeriaceae, are a well-studied group of fungi best known for the canker diseases they cause on woody hosts especially in stressed or damaged trees. Australian Plant Pathology herbaria contain many records for this group, but due to considerable taxonomic changes over the past decade, many of the species names have since been reclassified. In this article we used all published records with available sequence data of the Botryosphaeriaceae in Australia to examine the distribution and host range of these taxa. There are 24 genera encompassing 222 species in the Botryosphaeriaceae; 9 genera and 62 species have been recorded in Australia. Some genera such as Neoscytalidium are only found in warm, humid climates while Dothiorella species are more common in temperate climates. There were species, such as Lasiodiplodia theobromae , Neofusicoccum parvum and Botryosphaeria dothidea , which had a wide host range with many records. However, there were also several species found only in one location on a single host. While systematic data collection is still required, the information presented here provides a baseline of species present in Australia and will underpin future studies into this group of important pathogens.

Black root rot is a severe disease of young avocado trees in Australia causing black necrotic roots, tree stunting, and leaf drop prior to tree death. Nectriaceous fungi (Nectriaceae, Hypocreales), are commonly isolated from symptomatic roots. This research tested the pathogenicity of 19 isolates from Calonectria, Cylindrocladiella, Dactylonectria, Gliocladiopsis, and Ilyonectria, spp. collected from young avocado trees and other hosts. Glasshouse pathogenicity tests with 'Reed' avocado (Persea americana) seedlings confirmed that Calonectria ilicicola is a severe pathogen of avocado, causing stunting, wilting, and seedling death within 5 weeks of inoculation. Isolates of C. ilicicola from peanut, papaya, and custard apple were also shown to be aggressive pathogens of avocado, demonstrating a broad host range. An isolate of a Calonectria sp. from blueberry and avocado isolates of Dactylonectria macrodidyma, D. novozelandica, D. pauciseptata, and D. anthuriicola caused significant root rot but not stunting within 5 to 9 weeks of inoculation. An isolate of an Ilyonectria sp. from grapevine closely related to Ilyonectria liriodendri, and avocado isolates of Cylindrocladiella pseudoinfestans, Gliocladiopsis peggii, and an Ilyonectria sp. were not pathogenic to avocado.

Root rot of avocado (Persea americana) is an important disease in seedling nurseries as well as in the field in eastern and southern Australia. During an investigation into the causal organisms of avocado root rot, 19 isolates of Gliocladiopsis were obtained from necrotic lesions on avocado roots and examined by morphology and comparison of DNA sequences from three gene loci (the internal transcribed spacer region of the nuclear rDNA, Histone H3 and β-tubulin). Three new species of Gliocladiopsis are described as a result of phylogenetic analysis of these data. One of the new species, G. peggii, formed a monophyletic group that may represent an unresolved species complex as it contained a polytomy that included a well-supported clade comprising two subclades. Gliocladiopsis peggii is sister to G. mexicana, which is known from soil in Mexico. The remaining two new species, G. whileyi and G. forsbergii, formed a clade sister to G. curvata, which is known from Ecuador, Indonesia and New Zealand.

This greenhouse study investigated the efficacy of acibenzolar-S-methyl (Bion®) treatment of lower leaves of passionfruit, (Passiflora edulis f. sp. flavicarpa), on Passionfruit woodiness disease and activities of two pathogenesis-related proteins, chitinase and β-1,3-glucanase after inoculation with passionfruit woodiness virus (PWV). All Bion® concentrations reduced disease symptoms, but the concentration of 0.025 g active ingredient (a.i.)/l was the most effective, reducing disease severity in systemic leaves by 23, 29 and 30 % compared with water-treated controls at 30, 40 and 50 days post inoculation (dpi) with PWV, respectively. Correspondingly, relative virus concentration as determined by DAS-ELISA in the upper, untreated leaves (new growth) above the site of inoculation at 50 dpi was reduced by 17 and 22 % in plants treated with 0.025 and 0.05 g a.i./l, respectively. Bion® treatment and subsequent inoculation with PWV increased chitinase and β-1,3-glucanase activities in the new leaves above the site of inoculation at 30 dpi with PWV. It was concluded that optimal protective Bion® treatment concentrations were 0.025 and 0.05 g a.i./l.

Black root rot is a severe disease of nursery avocado trees and orchard transplants caused by soilborne fungal pathogens in the Nectriaceae family. The genera reported to be associated with black root rot are Calonectria, Cylindrocladiella, Dactylonectria, Gliocladiopsis and Ilyonectria. These genera have not been widely studied in avocado, although the disease causes significant commercial loss, with symptoms including black, rotten roots; tree stunting; leaf wilt; and rapid tree decline and death. This PhD research aims to i) identify the nectriaceous fungal species found in avocado roots in Australia, using morphological studies and molecular phylogenetic analyses of fungal gene sequences; ii) to perform pathogenicity tests on avocado seedlings and fruit to determine the pathogenic species; iii) to investigate whether the pathogens produce phytotoxic exudates which induce and facilitate disease symptom development; iv) and to use the generated gene sequence data to develop a molecular diagnostic for rapidly detecting the pathogens.Fungal isolates were obtained from symptomatic roots from sick and healthy avocado trees, nursery stock, young orchard transplants and mature established orchard trees from all growing regions in Australia, and from other host species. Bayesian inference and Maximum likelihood phylogenetic analyses of concatenated ITS, β-tubulin and histone H3 gene loci were used to identify and classify 153 Nectriaceae isolates in the genera Calonectria, Cylindrocladiella, Dactylonectria, Gliocladiopsis, Ilyonectria and Mariannaea. Three new species of Gliocladiopsis were identified and described as G. peggii, G. forsbergii and G. whileyi in a taxonomic study focusing on this genus. Comprehensive phylogenetic analyses also revealed additional new species and species complexes in each genus. Fungal species associated with black root rot of avocado in Australia was shown to include: species complexes of Calonectria ilicicola, Dactylonectria macrodidyma, D. anthuriicola, D. vitis, D. pauciseptata and Gliocladiopsis peggii, all containing putative novel species; D. novozelandica, putative novel and unresolved species of Ilyonectria, unresolved Gliocladiopsis sp., G. forsbergii, G. whileyi, Cylindrocladiella pseudoinfestans, Mariannaea humicola and a putative novel species of Mariannaea.Glasshouse pathogenicity tests confirmed fungal isolates of Calonectria ilicicola from avocado, papaya, peanut and custard apple caused significant root rot in avocado cv. Reed seedlings within 5 weeks of inoculation, inferring potential disease transmission between different hosts. These isolates also caused significant tree stunting, wilted leaves and seedling death in this time period. A Calonectria sp. isolate from blueberry was also found to cause black root rot in avocado seedlings but not stunting. New disease records in Australia were established for Dactylonectria macrodidyma, D. novozelandica, D. pauciseptata and D. anthuriicola as pathogens of avocado, causing black root rot in glasshouse experiments within 9 weeks of inoculation; however, stunting was not observed. Avocado isolates of Cylindrocladiella pseudoinfestans, Gliocladiopsis peggii and Ilyonectria sp., and a grapevine isolate of Ilyonectria sp. were confirmed as non-pathogenic to avocado seedlings. Pathogenicity experiments on avocado cv. Hass fruit showed necrotic lesions developed on fruit skin at mechanically wounded sites inoculated with Ca. ilicicola isolates from avocado and papaya, and D. macrodidyma and Ilyonectria sp. isolates from avocado. However only Ca. ilicicola isolates from avocado were able to infect and cause necrosis at non-wounded sites. Fungal culture filtrates (CF) of Calonectria ilicicola and Dactylonectria macrodidyma grown in potato dextrose broth (PDB) were tested for the ability to induce and facilitate phytotoxic symptom development in tomato seedling model plants, and in avocado cv. Reed leaves and fruit. Purified Brefeldin A (BFA), a known phytotoxin produced by Ilyonectria and Dactylonectria spp., was also tested for symptom development in tomato seedlings. Tomato seedlings treated with BFA or CF of Ca. ilicicola developed significant leaf wilt, chlorosis and necrosis by 12 days post treatment, compared to plants treated with water. However, seedlings treated with D. macrodidyma CF were not significantly different to plants treated with unamended PDB. Symptoms in plants treated with Ca. ilicicola CF were statistically similar to those treated with BFA, and plants of both treatments were developed symptoms more rapidly and severely than seedlings treated with D. macrodidyma CF or PDB. Glasshouse and in vitro trials on avocado leaves were inconclusive. Necrotic lesions developed on avocado fruit cv. Reed regardless of treatment and there was inconclusive evidence to suggest the facilitation of Ca. ilicicola or D. macrodidyma CF on disease symptom development by nectriaceous fungi. The necrotic lesions on fruit singularly treated with Ca. ilicicola CF were significantly larger than lesions formed on fruit treated with unamended media, suggesting that nectriaceous fungal exudates may potentially facilitate the exacerbation of post-harvest disease.The pathogens Calonectria iliciciola and Dactylonectria macrodidyma were selected for the development of a rapid, loop-mediated isothermal amplification (LAMP) diagnostic test. The collection of nectriaceous fungal DNA sequence data from three gene loci enabled the identification of candidate genes containing unique fixed nucleotides for specific species detection. Histone H3 was selected for detecting D. macrodidyma and β-tubulin was selected for detecting Ca. ilicicola. Species-specific LAMP primers were designed from the unique fixed nucleotides of those genes. The LAMP diagnostic was sensitive and specific to 0.01ng/µl of fungal DNA and could detect fungal mycelia of Ca. ilicicola within 12–25 minutes and D. macrodidyma within 13–28 minutes. Detection was significantly faster when tested with pure DNA, with Ca. ilicicola detected within 11 minutes on average and D. macrodidyma detected within 12–20 minutes. Calonectria ilicicola was successfully detected in necrotic avocado roots of cv. Reed seedlings previously inoculated in the glasshouse.The PhD study has significantly expanded knowledge on the diversity of nectriaceous fungal species associated with avocado, identified pathogens which cause black root rot disease in Australian avocado trees, demonstrated a potential role of phytotoxic exudates in pathogenicity, and contributed towards improving disease management in the global avocado industry through the development of a rapid molecular diagnostic test.