Assessing the Pathogenicity of Berkeleyomyces rouxiae and Fusarium oxysporum f. sp. vasinfectum on Cotton (Gossypium hirsutum) Using a Rapid and Robust Seedling Screening Method.

Cotton (Gossypium spp.) is the most important fibre crop worldwide. Black root rot and Fusarium wilt are two major diseases of cotton caused by soil-borne Berkeleyomyces rouxiae and Fusarium oxysporum f. sp. vasinfectum (Fov), respectively. Phenotyping plant symptoms caused by soil-borne pathogens has always been a challenge. To increase the uniformity of infection, we adapted a seedling screening method that directly uses liquid cultures to inoculate the plant roots and the soil. Four isolates, each of B. rouxiae and Fov, were collected from cotton fields in Australia and were characterised for virulence on cotton under controlled plant growth conditions. While the identities of all four B. rouxiae isolates were confirmed by multilocus sequencing, only two of them were found to be pathogenic on cotton, suggesting variability in the ability of isolates of this species to cause disease. The four Fov isolates were phylogenetically clustered together with the other Australian Fov isolates and displayed both external and internal symptoms characteristic of Fusarium wilt on cotton plants. Furthermore, the isolates appeared to induce varied levels of plant disease severity indicating differences in their virulence on cotton. To contrast the virulence of the Fov isolates, four putatively non-pathogenic Fusarium oxysporum (Fo) isolates collected from cotton seedlings exhibiting atypical wilt symptoms were assessed for their ability to colonise cotton host. Despite the absence of Secreted in Xylem genes (SIX6, SIX11, SIX13 and SIX14) characteristic of Fov, all four Fo isolates retained the ability to colonise cotton and induce wilt symptoms. This suggests that slightly virulent strains of Fo may contribute to the overall occurrence of Fusarium wilt in cotton fields. Findings from this study will allow better distinction to be made between plant pathogens and endophytes and allow fungal effectors underpinning pathogenicity to be explored.

Fluorescently Tagged Verticillium dahliae to Understand the Infection Process on Cotton (Gossypium hirsutum) and Weed Plant Species.

Verticillium wilt is a soil-borne disease caused by distinct vegetative compatibility groups (VCG) of the fungus Verticillium dahliae. Defoliating (VCG 1A) and non-defoliating (VCG 2A) pathotypes of V. dahliae have contributed to yield losses of cotton production in Australia. To study the virulence and the infection process of V. dahliae on cotton, two isolates, one representing each VCG, have been transformed with fluorescent protein genes. The transformants maintained their ability to infect the host, and both strains were observed to move through the plant vasculature to induce wilt symptoms. Furthermore, virulence testing suggests that the cotton V. dahliae strains can endophytically colonise common weed plant species found in the Australian landscape, and that is contrasted by their ability to infect and colonise native tobacco plants. The fluorescently labelled strains of V. dahliae not only allowed us to gain a thorough understanding of the infection process but also provided a method to rapidly identify recovered isolates from host colonisation studies.

Surveillance, Diversity and Vegetative Compatibility Groups of Fusarium oxysporum f. sp. vasinfectum Collected in Cotton Fields in Australia (2017 to 2022)

Cotton (Gossypium hirsutum) is a billion-dollar crop in regional New South Wales (NSW) and Queensland, Australia. Fusarium wilt (FW) caused by Fusarium oxysporum f. sp. vasinfectum (Fov) is an economically important disease. Initial disease losses of up to 90% when the disease was first detected resulted in fields being taken out of cotton production. The disease is now well-managed due to the adoption of highly resistant varieties. However, annual disease surveys recently revealed that the disease dynamic has changed in the past few seasons. With relatively mild and wet weather conditions during the 2021/22 growing season, FW was detected in eight surveyed valleys in NSW and Queensland, with the disease incidence as high as 44.5% and 98.5% in individual fields in early and late seasons, respectively. Fov is genetically distinct and evolved from local Fusarium oxysporum strains. Additionally, the pathogen was reported to evolve rapidly under continuous cotton cropping pressure. However, our knowledge of the genetic composition of the prevailing population is limited. Sequences of the translation elongation factor alpha 1 (TEF1) revealed that 94% of Fusarium isolates recovered from FW-infected cotton were clustered together with known Australian Fov and relatively distant related to overseas Fov races. All these isolates, except for nine, were further confirmed positive with a specific marker based on the Secreted in Xylem 6 (SIX6) effector gene. Vegetative compatibility group (VCG) analyses of 166 arbitrarily selected isolates revealed a predominance of VCG01111. There was only one detection of VCG01112 in the Border Rivers valley where it was first described. In this study, the exotic Californian Fov race 4 strain was not detected using a specific marker based on the unique Tfo1 insertion in the phosphate (PHO) gene. This study indicated that the prevalence and abundance of Fov across NSW and Queensland in the past five seasons was probably independent of its genetic diversity.

Co-Occurrence of Defoliating and Non-Defoliating Pathotypes of Verticillium Dahliae in Field-Grown Cotton Plants in New South Wales, Australia.

Verticillium wilt (VW) is a major constraint to cotton production in Australia and worldwide. The disease is caused by a soilborne fungus, Verticillium dahliae, a highly virulent pathogen on cotton. Commonly, V. dahliae is designated into two pathotypes: defoliating (D) and non-defoliating (ND), based on induced symptoms. In the previous two survey seasons between 2017 and 2019, stems with suspected VW were sampled for the confirmation of presence and distribution of D and ND pathotypes across New South Wales (NSW), Australia. A total of 151 and 84 VW-suspected stems sampled from the 2017/18 and 2018/19 seasons, respectively, were subjected to pathogen isolation. Of these, 94 and 57 stems were positive for V. dahliae; and 18 and 20 stems sampled respectively from the two seasons yielded the D pathotype isolates. Two stems from the 2017/18 season and one stem from 2018/19 season yielded both D and ND pathotype isolates. We also successfully demonstrated the co-infection of both pathotypes in pot trials, which was driven predominantly by either of the pathotypes, and appeared independent on vegetative growth, fecundity and spore germination traits. Our study is the first report of the natural co-occurrence of both D and ND pathotypes in same field-grown cotton plants in NSW, to which a challenge to the disease management will be discussed.

Development of Three Loop-Mediated Isothermal Amplification (LAMP) Assays for the Rapid Detection of Calonectria ilicicola, Dactylonectria macrodidyma, and the Dactylonectria Genus in Avocado Roots.

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 ß-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.

Effects of Silicon Amendment on Soilborne and Fruit Diseases of Avocado.

The effects of silicon (Si) amendment have been studied in several plant/pathogen interactions; however, studies in horticultural tree crops are limited. Effects of amendment with soluble potassium silicate (AgSil®32, approximately 30% available Si), or milled cement building board by-products (Mineral Mulch (MM) or Mineral Dust (MD), containing 5% available Si) were investigated in field and greenhouse trials with avocado. Orchard soil drench applications with potassium silicate improved yield and quality of fruit, but visual health of trees declining from Phytophthora root rot (PRR) was not affected. Orchard spray or trunk injection applications with potassium silicate were ineffective. Amendment of potting mix with MM and MD reduced root necrosis of avocado seedlings after inoculation with Calonectria ilicicola, an aggressive soilborne pathogen causing black root rot. Application of MM to mature orchard trees declining with PRR had a beneficial effect on visual tree health, and Si accumulation in leaves and fruit peel, after only 10 months. Products that deliver available Si consistently for uptake are likely to be most successful in perennial tree crops.