The catalase-peroxidase PiCP1 plays a critical role in abiotic stress resistance, pathogenicity and asexual structure development in Phytophthora infestans.

Catalase-peroxidase is a heme oxidoreductase widely distributed in bacteria and lower eukaryotes. In this study, we identified a catalase-peroxidase PiCP1 (PITG_05579) in Phytophthora infestans. PiCP1 had catalase/peroxidase and secretion activities and was highly expressed in sporangia and upregulated in response to oxidative and heat stresses. Compared with wild type, PiCP1-silenced transformants (STs) had decreased catalase activity, reduced oxidant stress resistance and damped cell wall integrity. In contrast, PiCP1-overexpression transformants (OTs) demonstrated increased tolerance to abiotic stresses and induced the upregulation of PR genes in the host salicylic acid pathway. The high concentration of PiCP1 can also induced callose deposition in plant tissue. Importantly, both STs and OTs have severely reduced sporangia formation and zoospore releasing rate, but the sporangia germination rate and type varied depending on environmental conditions. Comparative sequence analyses show that catalase-peroxidases are broadly distributed and highly conserved among soil-borne plant parasitic oomycetes, but not in freshwater-inhabiting or strictly plants-inhabiting oomycetes. In addition, we found that silencing PiCP1 downregulated the expression of PiCAT2. These results revealed the important roles of PiCP1 in abiotic stress resistance, pathogenicity and in regulating asexual structure development in response to environmental change. Our findings provide new insights into catalase-peroxidase functions in eukaryotic pathogens.

Bacterial and fungal communities within and among geographic samples of the hemp pest Psylliodes attenuata from China.

The hemp flea beetle Psylliodes attenuata (Coleoptera: Chrysomelidae: Psylliodes) is a common pest of Cannabis sativa, including cultivars of both medicinal marijuana and industrial hemp. Both the larval and adult stages of this beetle can cause significant damages to C. sativa, resulting in substantial crop losses. At present, little is known about the bacterial and fungal community diversity among populations of this pest insect. In the present study, we obtained P. attenuata samples from nine field sites representing broad industrial hemp productions in China and analyzed their microbial communities using DNA metabarcoding. Bacterial sequences of all the samples were assigned to 3728 OTUs, which belonged to 45 phyla, 1058 genera and 1960 known species. The most common genera were Rickettsia, Wolbachia, and Candidatus_Brownia. Fungal sequences of all the samples were assigned to 910 OTUs, which belonged to 9 phyla, 308 genera and 464 known species. The most common fungal genera were Cladosporium, Cutaneotrichosporon, and Aspergillus. Principal coordinate analysis revealed a significant difference in the bacterial and fungal community structure among the nine P. attenuata populations. Understanding the microbial symbionts may provide clues to help develop potential biocontrol techniques against this pest.

Genetic Diversity and Population Structure of Fusarium commune Causing Strawberry Root Rot in Southcentral China.

Strawberry plants and fruits are vulnerable to infections by a broad range of pathogens and pests. However, knowledge about the epidemiology of pathogens causing strawberry diseases is limited. In this study, we analyzed Fusarium commune, a major fungal pathogen causing strawberry root rot, from diseased strawberry root tissues in southcentral China. A total of 354 isolates were obtained from 11 locations that spanned about 700 km from both south to north and east to west. Multilocus genotypes of all isolates were obtained using seven polymorphic simple sequence repeat markers developed in this study. Our analyses revealed significant genetic diversity within each of the 11 local populations of F. commune. STRUCTURE analysis revealed that the optimal number of genetic populations for the 354 strains was two, with most local geographic populations containing isolates in both genetic clusters. Interestingly, many isolates showed allelic ancestry to both genetic clusters, consistent with recent hybridization between the two genetic clusters. In addition, though alleles and genotypes were frequently shared among local populations, statistically significant genetic differentiations were found among the local populations. However, the observed F. commune population genetic distances were not correlated with geographic distances. Together, our analyses suggest that populations of F. commune causing strawberry root rot are likely endemic to southcentral China, with each local population containing shared and unique genetic elements. Though the observed gene flow among geographic regions was relatively low, human activities will likely accelerate pathogen dispersals, resulting in the generation of new genotypes through mating and recombination.

Development of a Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Phytopythium vexans.

Brown root rot caused by Phytopythium vexans is a new destructive root disease on many plants such as Gingko, Citrus, kiwifruit, and ramie. The establishment of loop-mediated isothermal amplification (LAMP) technology for detecting P. vexans can help monitor and control brown root rot quickly, efficiently, and accurately. LAMP technology is known for its simplicity, sensitivity, and speed; and it does not require any specialized equipment - a water bath or a thermoblock is sufficient for isothermal amplifications. LAMP products can be visualized by using hydroxy naphthol blue (HNB) dye or agarose gel electrophoresis. In this study, by searching and comparing the internal transcribed spacer (ITS) sequences of P. vexans and the related species in oomycete genera Pythium, Phytopythium, and Phytophthora, we designed specific primers targeting the ITS gene region of P. vexans. Using HNB dye, we established a LAMP technique for rapid detection of P. vexans by visible color change. In addition, we optimized the protocol to enhance both sensitivity and specificity for P. vexans detection. Under the optimized condition, our protocol based on LAMP technology could detect as low as 24 copies of the P. vexans genomic DNA, which is ∼100 times more sensitive than conventional PCR. This method can successfully detect P. vexans using cell suspensions from P. vexans - infected ramie root tissues.

Linking Plant Secondary Metabolites and Plant Microbiomes: A Review.

Plant secondary metabolites (PSMs) play many roles including defense against pathogens, pests, and herbivores; response to environmental stresses, and mediating organismal interactions. Similarly, plant microbiomes participate in many of the above-mentioned processes directly or indirectly by regulating plant metabolism. Studies have shown that plants can influence their microbiome by secreting various metabolites and, in turn, the microbiome may also impact the metabolome of the host plant. However, not much is known about the communications between the interacting partners to impact their phenotypic changes. In this article, we review the patterns and potential underlying mechanisms of interactions between PSMs and plant microbiomes. We describe the recent developments in analytical approaches and methods in this field. The applications of these new methods and approaches have increased our understanding of the relationships between PSMs and plant microbiomes. Though the current studies have primarily focused on model organisms, the methods and results obtained so far should help future studies of agriculturally important plants and facilitate the development of methods to manipulate PSMs-microbiome interactions with predictive outcomes for sustainable crop productions.

First report of Fusarium fujikuroi causing bulb rot on Lilium lancifolium in China.

Lilium lancifolium Thunb., commonly known as Juandan lily and tiger lily, is widely cultivated in China for its edible bulbs and medicinal properties, with a commercial value worth of ~RMB 6 billion Yuan per year. Bulb rot is an increasingly common disease on L. lancifolium, significantly impacting both the quantity and quality of the main product, the scaled bulbs. Typically, the causal pathogens invade the plant through wounds in the root or the ends of the bulb, causing the roots and bulb to brown and rot, which can eventually lead to stem wilt and death of the whole plants. During pathogenesis, the infected bulbs typically turn from white to brown, with sunken lesions and later the scales flaking off from the base of the bulb (Figure 1A and 1B). Plants growing from infected bulbs are generally short, with discolored leaves, wilting, and death at an early stage. Bulb rot is commonly observed in fields with excess water and a history of continuous Juandan lily cultivation. For this study, wilted L. lancifolium plants with rotted bulbs were collected from Longshan in Hunan, Enshi in Hubei, Yixing in Jiangsu, and Lu'an in Anhui in 2018 and 2019. Infected bulbs were surface sterilized with 75% ethanol for 30 seconds, followed by disinfection with 2% sodium hypochlorite for 5 minutes, and then rinsing with sterile water three times. The surface-sterilized tissue was divided into small pieces of 0.5 × 0.5 cm in size, placed on potato dextrose agar (PDA) medium containing 50 mg/l streptomycin sulfate, and incubated at 25℃. Mycelia growing from diseased tissues were sub-cultured onto fresh PDA medium to obtain pure culture, which formed dense white hyphae after a few days (Figure 1C and 1D). Colonies on PDA produced abundant condia about 15 days after subculturing. Microconidia were abundant, solitary, thin walled, hyaline, ovoid, 0 to 1 septate, with an average size of 6.1 × 2.6 µm (n=50) (Figure 1E). Macroconidia had a curved apical cell and foot-like basal cell with 3 to 5 septa, with an average size of 35.4 × 4.3 µm (n=30) (Figure 1E). No chlamydospore was observed. These morphological characteristics of the causal pathogen were similar to those of Fusarium spp. (Leslie et al., 2006). To identify the Fusarium isolates to species level, DNA fragments of the internal transcribed spacer (ITS) regions of the ribosomal RNA gene cluster, translation elongation factor subunit 1-alpha (TEF1-α), and RNA polymerase II subunit 2 (RPB2) genes were amplified using primers ITS1/ITS4, EF1/EF2, and 7cF/11aR respectively and sequenced (Choi et al. 2018; Jiang et al. 2018; Choi et al. 2017). BLAST analyses showed that the ITS (GenBank Accession No. MT549849), TEF1-α (GenBank Accession No. MT553348), and RPB2 (Accession No. MW201686) sequences of our isolates shared the highest sequence identities (98-100%) with those of F. fujikuroi reference strains in GenBank. A phylogenetic tree showing the relationship between one of our strains, S106, and those of the closely related species within the F. fujikuroi species complex was constructed by the maximum likelihood method using MEGA X (Kumar et al. 2018) (Figure 2). Based on the morphological characteristics and DNA sequences, the strains were identified as F. fujikuroi sensu stricto. We used two methods, an ex vivo assay using Juandan lily bulb scales and an in vivo assay using potted Juandan lily plants, to confirm pathogenicity for one representative F. fujikuroi strain from each of the four geographic regions to fulfill Koch's postulates (Bian et al. 2016; Zeng et al. 2019). In the ex vivo assay, actively growing mycelia on PDA plates were cut into 5mm diameter fungal blocks as inocula. To prepare healthy Juandan lily bulb scales as test tissues, healthy fresh scales were first surface sterilized using 75% alcohol for 30 seconds, followed by treatment of 2% sodium hypochlorite for 5 minutes, and then rinsed with sterile water 3 times. The scales were punctured with sterilized dissecting needles, the 5mm mycelial blocks containing the PDA medium were then inoculated on the punctured wound of the scales. Sterile PDA culture medium without mycelia was inoculated on the punctured wound as a negative control. After inoculation, Juandan lily scales were placed in sterile culture dishes with two layers of sterilized filter paper and 5ml of sterile water in each dish. Six Juandan lily scales were placed in each dish, with different treatments placed in different dishes, and the dishes were placed in an incubator in the dark at 25℃. After 10 days of incubation, we found that the F. fujikuroi-inoculated Juandan lily bulb scales showed disease symptoms (brownish lesion) similar to those in the original field collected infected bulb samples (Figure 1F). However, such symptoms were not observed in the negative control group. The pathogenicity test was performed 3 times for each isolate, each with six repeats. In the in vivo pathogenicity test using potted lily plants, we prepared actively growing cultures of our F. fujikuroi strains by incubating them in a liquid medium, the potato dextrose broth, for 3 days in a shaker-incubator at 25℃ and 180rpm. The asexual spores conidia from the fungal cultures were harvested by filtration through eight layers of sterile cheese clothes and with spore concentrations adjusted to 1×107 conidia per ml. Healthy Juandan lily bulbs were selected and one bulb was planted in each pot containing sterilized soil. Each pot was inoculated with 1ml conidia suspension, at the base soil where the bulbs were planted. The pots were placed in a growth chamber at 25℃ with a 12 h light and 12 h dark cycle. Symptoms similar to those observed in diseased bulbs in the field were observed, with symptoms at 30 days after inoculations shown in Figure 3. Specifically, most of the roots, bulb plate and scale tissues of Juandan lily plants inoculated with F. fujikuroi conidia were rotten and turned black, with few new roots. In addition, the infected plants showed stunted growth (Figure 3). In contrast, the uninoculated plants grew normally, with dense new roots and healthy-looking bulbs, and no rot symptom (Figure 3). The fungi were re-isolated from the infected Juandan lily tissues from both pathogenicity assays, following the procedures described above for isolating and identifying the fungal cultures from infected field samples. These re-isolated fungi were shown to have colony morphology and DNA sequences at the three loci identical to those of our inoculated F. fujikuroi strains. Several Fusarium species have been reported as pathogens of lily plants in China, including F. oxysporum, F. solani and F. tricinctum (Li, et al., 1995; Li, et al., 2013). In addition, F. redolens has been reported previously in ornamental lily in Ukraine (Zerova, 1940). Indeed, Fusarium moniliforme, one of the disused synonyms of F. fujikuroi (Seifert et al. 2003), has been reported as a causal agent for diseases in lily. However, it's now known that the originally defined F. fujikuroi sensu lato is in fact a large species complex consisting of over 60 recognized species, including F. fujikuroi sensu stricto (Moussa et al. 2017; Choi et al. 2018). In addition, there are over 100 species in the genus Lilium as well as many other species with their common names including the word "lily" but are not in the Lilium genus. To our knowledge, this is the first confirmed report of bulb rot of Juandan lily L. lancifolium caused by F. fujikuroi sensu stricto in China. Our result should help with future monitoring and control of Juandan lily diseases.

Sphinganine-Analog Mycotoxins (SAMs): Chemical Structures, Bioactivities, and Genetic Controls.

Sphinganine-analog mycotoxins (SAMs) including fumonisins and A. alternata f. sp. Lycopersici (AAL) toxins are a group of related mycotoxins produced by plant pathogenic fungi in the Fusarium genus and in Alternaria alternata f. sp. Lycopersici, respectively. SAMs have shown diverse cytotoxicity and phytotoxicity, causing adverse impacts on plants, animals, and humans, and are a destructive force to crop production worldwide. This review summarizes the structural diversity of SAMs and encapsulates the relationships between their structures and biological activities. The toxicity of SAMs on plants and animals is mainly attributed to their inhibitory activity against the ceramide biosynthesis enzyme, influencing the sphingolipid metabolism and causing programmed cell death. We also reviewed the detoxification methods against SAMs and how plants develop resistance to SAMs. Genetic and evolutionary analyses revealed that the FUM (fumonisins biosynthetic) gene cluster was responsible for fumonisin biosynthesis in Fusarium spp. Sequence comparisons among species within the genus Fusarium suggested that mutations and multiple horizontal gene transfers involving the FUM gene cluster were responsible for the interspecific difference in fumonisin synthesis. We finish by describing methods for monitoring and quantifying SAMs in food and agricultural products.

Genome Sequence Resource for the Ramie Oomycete Pathogen Phytopythium vexans HF1.

The oomycete Phytopythium vexans is a causative agent of patch canker, damping-off, and crown, stem, and root rot in many economically important plants. P. vexans HF1 was isolated in China, where it caused brown root rot of ramie, a fiber crop broadly cultivated in Asia. The genome of HF1 was sequenced by a combination of technologies producing short (Illumina HiSeq X) and long (PacBio RS) reads. The genome is 41.73 Mbp long, assembled into 44 contigs. It has a GC content of 58.17% and contains 13,051 predicted coding genes, including 1,461 putative virulence genes and 220 putative antimicrobial resistance genes. This genome sequence provides a resource for determining the molecular mechanisms of disease development in this pathosystem.

Mitochondrial COI Sequence Variations within and among Geographic Samples of the Hemp Pest Psylliodes attenuata from China.

The hemp flea beetle Psylliodes attenuata (Coleoptera: Chrysomelidae: Psylliodes) is a common pest of Cannabis sativa, including cultivars of both industrial hemp and medicinal marijuana. Both the larval and adult stages of this beetle can cause significant damages to C. sativa, resulting in substantial crop losses. At present, little is known about the populations of this pest, including its genetic diversity. In this study, we obtained 281 P. attenuata samples from nine field sites representing broad industrial hemp productions in China and analyzed their DNA sequences at the mitochondrial COI gene, the insect DNA barcode. Our analyses revealed a total of 48 haplotypes, with 28 being found only in one specimen each while the remaining 20 were shared by two or more specimens each. Of the 20 shared haplotypes, eight were shared among local populations often from far away locations, consistent with recent long-distance dispersals. However, the observed putative long-distance dispersals have not obscured the significant genetic differentiations among the regional populations from northeastern, eastern, central and southwestern China. Interestingly, haplotype network analyses suggest evidence for potential mitochondrial recombination in natural populations of this species. We briefly discuss the implications of our results on its evolution, center of diversity, route of spread, and pest management strategies in hemp fields.

Molecular Diagnostics and Detection of Oomycetes on Fiber Crops.

Fiber crops are an important group of economic plants. Traditionally cultivated for fiber, fiber crops have also become sources of other materials such as food, animal feed, cosmetics and medicine. Asia and America are the two main production areas of fiber crops in the world. However, oomycete diseases have become an important factor limiting their yield and quality, causing devastating consequences for the production of fiber crops in many regions. To effectively control oomycete pathogens and reduce their negative impacts on these crops, it is very important to have fast and accurate detection systems, especially in the early stages of infection. With the rapid development of molecular biology, the diagnosis of plant pathogens has progressed from relying on traditional morphological features to the increasing use of molecular methods. The objective of this paper was to review the current status of research on molecular diagnosis of oomycete pathogens on fiber crops. Our search of PubMed identified nearly 30 species or subspecies of oomycetes on fiber crops, among which the top three species were Phytophthora boehmeriae, Phytophthora nicotianae and Pythium ultimum. The gene regions that have been used for molecular identifications of these pathogens include the internal transcribed spacer (ITS) regions of the nuclear ribosomal RNA gene cluster, and genes coding for translation elongation factor 1α (EF-1α) and mitochondrial cytochrome c oxidase subunits I and II (Cox 1, Cox 2), etc. We summarize the molecular assays that have been used to identify these pathogens and discuss potential areas of future development for fast, specific, and accurate diagnosis of oomycetes on fiber crops.

Molecular Diagnostics and Pathogenesis of Fungal Pathogens on Bast Fiber Crops.

Bast fibers and products derived from them are undergoing a resurgence in demand in the global market. However, fungal diseases have become an important factor limiting their yield and quality, causing devastating consequences for the production of bast fiber crops in many parts of the world. Thus, there is a high demand for effective control and prevention strategies against fungal pathogens. Having rapid, specific, sensitive, and cost-effective tests that can be used for early and accurate diagnosis of disease agents is an essential step of such strategies. The objective of this study was to review the current status of research on molecular diagnosis of fungal pathogens on bast fiber crops. Our search of PubMed identified nearly 20 genera of fungal pathogens on bast fiber crops, among which the five most common genera were Colletotrichum, Pythium, Verticillium, Fusarium, and Golovinomyces. The gene regions that have been used for molecular identifications of these fungi include internal transcribed spacer (ITS), translation elongation factor 1-α (EF-1α), ß-tubulin, calmodulin (CAL), histone subunit 3 (H3), glyceraldehydes-3-phosphate dehydrogenase (GAPDH), etc. We summarize the molecular assays that have been used to identify these fungi and discuss potential areas of future development for fast, specific, and accurate diagnosis of fungal pathogens on bast fiber crops.

A proper PiCAT2 level is critical for sporulation, sporangium function, and pathogenicity of Phytophthora infestans.

Catalase is present in prokaryotic and eukaryotic organisms and is important for the protective effects of the antioxidant system against free radicals. Many studies have confirmed that catalase is required for the growth, development, and pathogenesis of bacteria, plants, animals, and fungi. However, there has been relatively little research on the catalases in oomycetes, which form an important group of fungus-like eukaryotes that produce zoosporangia. In this study, we detected two Phytophthora infestans genes encoding catalases, but only PiCAT2 exhibited catalase activity in the sporulation stage and was highly produced during asexual reproduction and in the late infection stage. Compared with the wild-type strain, the PiCAT2-silenced P. infestans transformants were more sensitive to abiotic stress, were less pathogenic, and had a lower colony expansion rate and lower PiMPK7, PiVPS1, and PiGPG1 expression levels. In contrast, the PiCAT2-overexpressed transformants were slightly less sensitive to abiotic stress. Interestingly, increasing and decreasing PiCAT2 expression from the normal level inhibited sporulation, germination, and infectivity, and down-regulated PiCdc14 expression, but up-regulated PiSDA1 expression. These results suggest that PiCAT2 is required for P. infestans mycelial growth, asexual reproduction, abiotic stress tolerance, and pathogenicity. However, a proper PiCAT2 level is critical for the formation and normal function of sporangia. Furthermore, PiCAT2 affects P. infestans sporangial formation and function, pathogenicity, and abiotic stress tolerance by regulating the expression of cell cycle-related genes (PiCdc14 and PiSDA1) and MAPK pathway genes. Our findings provide new insights into catalase functions in eukaryotic pathogens.