Department of Plant Pathology

Candidatus Liberibacter spp. are insect-vectored, fastidious, and vascular-limited phytopathogens. They are the presumptive causal agents of potato zebra chip, tomato vein clearing, and the devastating citrus greening disease worldwide. There is an urgent need to develop new strategies to control them. In this study, we characterized a dual-specificity serine/tyrosine phosphatase (STP) that is well conserved among thirty-three geographically diverse Candidatus Liberibacter spp. and strains that infect multiple Solanaceaea and citrus spp. The STP is expressed in infected plant tissues, localized at the plant cytosol and plasma membrane, and interferes with plant cell death responses. We employed an in silico target-based molecular modeling and ligand screen to identify two small molecules with high binding affinity to STP. Efficacy studies demonstrated that the two molecules can inhibit Candidatus Liberibacter spp. but not unrelated pathogens and confer plant disease tolerance. The inhibitors and strategies are promising means to control Candidatus Liberibacter spp.

Autophagy in eukaryotes functions to maintain homeostasis by degradation and recycling of long-lived and unwanted cellular materials. Autophagy plays important roles in pathogenicity of various fungal pathogens, suggesting that autophagy is a novel target for development of antifungal compounds. Here, we describe bioluminescence resonance energy transfer (BRET)-based high-throughput screening (HTS) strategy to identify compounds that inhibit fungal ATG4 cysteine protease-mediated cleavage of ATG8 that is critical for autophagosome formation. We identified ebselen (EB) and its analogs ebselen oxide (EO) and 2-(4-methylphenyl)-1,2-benzisothiazol-3(2H)-one (PT) as inhibitors of fungal pathogens Botrytis cinerea and Magnaporthe oryzae ATG4-mediated ATG8 processing. The EB and its analogs inhibit spore germination, hyphal development, and appressorium formation in Ascomycota pathogens, B. cinerea, M. oryzae, Sclerotinia sclerotiorum and Monilinia fructicola. Treatment with EB and its analogs significantly reduced fungal pathogenicity. Our findings provide molecular insights to develop the next generation of antifungal compounds by targeting autophagy in important fungal pathogens.

In the original publication [...].

The phyllosphere, or plant leaf surface, represents a microbial ecosystem of considerable size, holding extraordinary biodiversity and enormous potential for the discovery of new products, tools, and applications in biotechnology, agriculture, medicine, and elsewhere. This mini-review highlights the applied microbiology of the phyllosphere as an original field of study concerning itself with the genes, gene products, natural compounds, and traits that underlie phyllosphere-specific adaptations and services that have commercial and economic value for current or future innovation. Examples include plant-growth-promoting and disease-suppressive phyllobacteria, probiotics and fermented foods that support human health, as well as microbials that remedy foliar contamination with airborne pollutants, residual pesticides, or plastics. Phyllosphere microbes promote plant biomass conversion into compost, renewable energy, animal feed, or fiber. They produce foodstuffs such as thickening agents and sugar substitutes, industrial-grade biosurfactants, novel antibiotics and cancer drugs, as well as enzymes used as food additives or freezing agents. Furthermore, new developments in DNA sequence-based profiling of leaf-associated microbial communities allow for surveillance approaches in the context of food safety and security, for example, to detect enteric human pathogens on leafy greens, predict plant disease outbreaks, and intercept plant pathogens and pests on internationally traded goods. KEY POINTS: • Applied phyllosphere microbiology concerns leaf-specific adaptations for economic value • Phyllobioprospecting searches the phyllosphere microbiome for product development • Phyllobiomonitoring tracks phyllosphere microbial profiles for early risk detection.

BACKGROUND: Fungal plant pathogens have dynamic genomes that allow them to rapidly adapt to adverse conditions and overcome host resistance. One way by which this dynamic genome plasticity is expressed is through effector gene loss, which enables plant pathogens to overcome recognition by cognate resistance genes in the host. However, the exact nature of these loses remains elusive in many fungi. This includes the tomato pathogen Cladosporium fulvum, which is the first fungal plant pathogen from which avirulence (Avr) genes were ever cloned and in which loss of Avr genes is often reported as a means of overcoming recognition by cognate tomato Cf resistance genes. A recent near-complete reference genome assembly of C. fulvum isolate Race 5 revealed a compartmentalized genome architecture and the presence of an accessory chromosome, thereby creating a basis for studying genome plasticity in fungal plant pathogens and its impact on avirulence genes. RESULTS: Here, we obtained near-complete genome assemblies of four additional C. fulvum isolates. The genome assemblies had similar sizes (66.96 to 67.78 Mb), number of predicted genes (14,895 to 14,981), and estimated completeness (98.8 to 98.9%). Comparative analysis that included the genome of isolate Race 5 revealed high levels of synteny and colinearity, which extended to the density and distribution of repetitive elements and of repeat-induced point (RIP) mutations across homologous chromosomes. Nonetheless, structural variations, likely mediated by transposable elements and effecting the deletion of the avirulence genes Avr4E, Avr5, and Avr9, were also identified. The isolates further shared a core set of 13 chromosomes, but two accessory chromosomes were identified as well. Accessory chromosomes were significantly smaller in size, and one carried pseudogenized copies of two effector genes. Whole-genome alignments further revealed genomic islands of near-zero nucleotide diversity interspersed with islands of high nucleotide diversity that co-localized with repeat-rich regions. These regions were likely generated by RIP, which generally asymmetrically affected the genome of C. fulvum. CONCLUSIONS: Our results reveal new evolutionary aspects of the C. fulvum genome and provide new insights on the importance of genomic structural variations in overcoming host resistance in fungal plant pathogens.

Gene cloning in repeat-rich polyploid genomes remains challenging. Here, we describe a strategy for overcoming major bottlenecks in cloning of the powdery mildew resistance gene (R-gene) Pm69 derived from tetraploid wild emmer wheat. A conventional positional cloning approach was not effective owing to suppressed recombination. Chromosome sorting was compromised by insufficient purity. A Pm69 physical map, constructed by assembling Oxford Nanopore Technology (ONT) long-read genome sequences, revealed a rapidly evolving nucleotide-binding leucine-rich repeat (NLR) R-gene cluster with structural variations. A single candidate NLR was identified by anchoring RNA sequencing reads from susceptible mutants to ONT contigs and was validated by virus-induced gene silencing. Pm69 is likely a newly evolved NLR and was discovered in only one location across the wild emmer wheat distribution range in Israel. Pm69 was successfully introgressed into cultivated wheat, and a diagnostic molecular marker was used to accelerate its deployment and pyramiding with other R-genes.

Eucalyptus camaldulensis is a multifunctional tree and is globally used for the reclamation of problematic lands. Eucalyptus camaldulensis is prone to attack by a number of pathogens, but the most important threat is the Fusarium wilt (Fusarium oxysporum). Keeping in view the importance of E. camaldulensis and to manage this disease, five plant activators, i.e., salicylic acid (C7H6O3), benzoic acid (C7H6O2), citric acid (C6H8O7), dipotassium phosphate (K2HPO4), monopotassium phosphate (KH2PO4) and nutritional mixture namely Compound (NPK) and nutriotop (Fe, Zn, Cu, B, Mn) were evaluated in the Fusarium infested field under RCBD in the Research Area, Department of Forestry and Range Management, University of Agriculture, Faisalabad (UAF). Among plant activators, salicylic acid and a combination of compound + nutriotop exhibited the lowest disease incidence and enhanced fresh and dry weight of leaves compared to other treatments and control. Results of the environmental study indicated maximum disease incidence between 35-40 °C (max. T), 6-25 °C (mini. T), 70-80% relative humidity and 1.5-2.5 km/h wind speed while pan evaporation expressed weak correlation with disease development. It was concluded that Fusarium wilt of Eucalyptus camaldulensis could be managed through activation of the basal defense system of the host plant with provision of salicylic acid and balanced nutrition by considering environmental factors. Recent exploration is expected to be helpful for future research efforts on epidemiology and ecologically sound intervention of Fusarium wilt of Eucalyptus camaldulensis.

We sequenced and comprehensively analysed the genomic architecture of 98 fluorescent pseudomonads isolated from different symptomatic and asymptomatic tissues of almond and a few other Prunus spp. Phylogenomic analyses, genome mining, field pathogenicity tests, and in vitro ice nucleation and antibiotic sensitivity tests were integrated to improve knowledge of the biology and management of bacterial blast and bacterial canker of almond. We identified Pseudomonas syringae pv. syringae, P. cerasi, and P. viridiflava as almond canker pathogens. P. syringae pv. syringae caused both canker and foliar (blast) symptoms. In contrast, P. cerasi and P. viridiflava only caused cankers, and P. viridiflava appeared to be a weak pathogen of almond. Isolates belonging to P. syringae pv. syringae were the most frequently isolated among the pathogenic species/pathovars, composing 75% of all pathogenic isolates. P. cerasi and P. viridiflava isolates composed 8.3 and 16.7% of the pathogenic isolates, respectively. Laboratory leaf infiltration bioassays produced results distinct from experiments in the field with both P. cerasi and P. syringae pv. syringae, causing significant necrosis and browning of detached leaves, whereas P. viridiflava conferred moderate effects. Genome mining revealed the absence of key epiphytic fitness-related genes in P. cerasi and P. viridiflava genomic sequences, which could explain the contrasting field and laboratory bioassay results. P. syringae pv. syringae and P. cerasi isolates harboured the ice nucleation protein, which correlated with the ice nucleation phenotype. Results of sensitivity tests to copper and kasugamycin showed a strong linkage to putative resistance genes. Isolates harbouring the ctpV gene showed resistance to copper up to 600 μg/ml. In contrast, isolates without the ctpV gene could not grow on nutrient agar amended with 200 μg/ml copper, suggesting ctpV can be used to phenotype copper resistance. All isolates were sensitive to kasugamycin at the label-recommended rate of 100μg/ml.

While quinones are essential for respiratory microorganisms, their importance for microbes that rely on fermentation metabolism is not understood. This gap in knowledge hinders our understanding of anaerobic microbial habitats, such in mammalian digestive tracts and fermented foods. We show that Lactiplantibacillus plantarum, a model fermentative lactic acid bacteria species abundant in human, animal, and insect microbiomes and fermented foods, uses multiple exogenous, environmental quinones as electron shuttles for a hybrid metabolism involving EET. Interestingly, quinones both stimulate this metabolism as well as cause oxidative stress when extracellular electron acceptors are absent. We also found that quinone-producing, lactic acid bacteria species commonly enriched together with L. plantarum in food fermentations accelerate L. plantarum growth and medium acidification through a mainly quinone- and EET-dependent mechanism. Thus, our work provides evidence of quinone cross-feeding as a key ecological feature of anaerobic microbial habitats.

Diaphorina citri is the hemipteran pest and vector of a devastating bacterial pathogen of citrus worldwide. In addition to the two core bacterial endosymbionts of D. citri, Candidatus Carsonella ruddii and Candidatus Profftella armatura, the genome of a novel endosymbiont and as of yet undescribed microbe was discovered in a Hawaiian D. citri population through deep sequencing of multiple D. citri populations. Found to be closely related to the genus Asaia in the family Acetobacteraceae by 16S rRNA gene sequence analysis, it forms a sister clade along with other insect-associated 16S rRNA gene sequences from uncultured bacterium found associated with Aedes koreicus and Sogatella furcifera. Multilocus sequence analysis confirmed the phylogenetic placement sister to the Asaia clade. Despite the culturable Asaia clade being the closest phylogenetic neighbour, attempts to culture this newly identified bacterial endosymbiont were unsuccessful. On the basis of these distinct genetic differences, the novel endosymbiont is proposed to be classified into a candidate genus and species 'Candidatus Kirkpatrickella diaphorinae'. The full genome was deposited in GenBank (accession number CP107052; prokaryotic 16S rRNA OP600170).