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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.

The demand for reliable methods for the quantification of intracellular bacteria is growing. Among modern methods such as PCR and flow cytometry, traditional methods including colony forming unit assay and immune-fluorescence are still the two most commonly techniques worldwide. In colony forming unit assay, there are variations among publications, making data results inconsistent across studies. The aim of this paper is to evaluate available techniques and develop improved protocols for the quantification of intracellular Listeria monocytogenes (LM) in vitro infection assay. This study has suggested different uptake time for phagocytic and non-phagocytic cells. Specifically, uptake time was determined at 0.5 hour after infection for RAW264.7 macrophages and 2 hours for L929 fibroblast host cells. To efficiently remove extracellular bacteria during infection period, gentamicin at high and low concentrations was used during the infection assay. High concentration of gentamicin was used to kill extracellular bacteria while low concentration of gentamicin was used to prevent secondary infection of host cells during the infection period. To obtain a more accurate number of alive LM from a large scale experiment, phosphate-buffered saline/PBS should be used rather than mili-Q (mQ) water to lyse the host cell as mQ water can kill additional bacteria unexpectedly. In immune-fluorescence, LM can be visualized by using either the LM expressing green fluorescence protein (GFP) or antibody against LM. To observed GFP signal, cells should be fixed with paraformaldehyde as methanol will rapidly dim the GFP signal. Findings from this study will benefit researchers engaged in both basic cell biology and infectious diseases.

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