De Novo long-read assembly and annotation for genomes of two isolates of Berkeleyomyces rouxiae

Lepidopteran species are mostly pests, causing serious annual economic losses. High-quality genome sequencing and assembly uncover the genetic foundation of pest occurrence and provide guidance for pest control measures. Long-read sequencing technology and assembly algorithm advances have improved the ability to timeously produce high-quality genomes. Lepidoptera includes a wide variety of insects with high genetic diversity and heterozygosity. Therefore, the selection of an appropriate sequencing and assembly strategy to obtain high-quality genomic information is urgently needed. This research used silkworm as a model to test genome sequencing and assembly through high-coverage datasets by de novo assemblies. We report the first nearly complete telomere-to-telomere reference genome of silkworm Bombyx mori (P50T strain) produced by Pacific Biosciences (PacBio) HiFi sequencing, and highly contiguous and complete genome assemblies of two other silkworm strains by Oxford Nanopore Technologies (ONT) or PacBio continuous long-reads (CLR) that were unrepresented in the public database. Assembly quality was evaluated by use of BUSCO, Inspector, and EagleC. It is necessary to choose an appropriate assembler for draft genome construction, especially for low-depth datasets. For PacBio CLR and ONT sequencing, NextDenovo is superior. For PacBio HiFi sequencing, hifiasm is better. Quality assessment is essential for genome assembly and can provide better and more accurate results. For chromosome-level high-quality genome construction, we recommend using 3D-DNA with EagleC evaluation. Our study references how to obtain and evaluate high-quality genome assemblies, and is a resource for biological control, comparative genomics, and evolutionary studies of Lepidopteran pests and related species.

SummarySeveral studies were carried out to investigate the soil microbial components involved in suppressing strawberry black rot root which occurs throughout the Italian strawberry growing region. Quantitative and qualitative evaluation of fungi involved in black root rot were combined with several soil microbial parameters involved in soil suppressiveness towards black root rot agents. The first survey, carried out in an intensively cultivated area of northern Italy, identified Rhizoctonia spp. as the main root pathogen together with several typical weak pathogens belonging to the well‐known black rot root complex of strawberry crop: Cylindrocarpondestructans, Fusarium oxysporum, F. solani, Pestalotia longiseta and others. The root colonisation frequency of strawberry plants increased strongly from autumn to spring at harvesting stage. Rhizoctonia spp. were the only pathogens which followed the rising trend of root colonisation with relative frequency; all the weak pathogens of strawberry black root rot complex did not vary their frequency. Only non‐pathogenic fungi decreased from autumn to spring when at least 60% of colonising fungi were represented by Rhizoctonia. These data suggested that the late vegetative stage was the best time to record the soil inoculum of root rot agents in strawberry using root infection frequency as a parameter of soil health. A further study was performed in two fields, chosen for their common soil texture and pH, but with significant differences in previous soil management: one (ALSIA) had been subjected to strawberry monoculture without organic input for several years; the other (CIF) has been managed according to a 4‐year crop rotation and high organic input. In this study Pythium artificially inoculated was adopted as an indicator for the behaviour of saprophytically living pathogens in bulk soil. Pythium showed a sharp, different response after inoculation in bulk soil from the two soil systems evaluated. Pythium was suppressed only in the CIF field where the highest levels of total fungi and fluorescent bacteria and highest variability were observed. The suppressiveness conditions towards Pythium, observed in the CIF and absent in the ALSIA field, corresponded with the root infection frequency recorded at the late vegetative stage on strawberry plants grown in the two fields: strawberry plants from the CIF field showed lower root colonisation frequency and higher variability than that recorded on those coming from the ALSIA field.

Sclerotinia sclerotiorum is a broad host range necrotrophic fungal pathogen, which causes disease on many economically important crop species. S. sclerotiorum has been shown to secrete small effector proteins to kill host cells and acquire nutrients. We set out to discover novel necrosis‐inducing effectors and characterize their activity using transient expression in Nicotiana benthamiana leaves. Five intracellular necrosis‐inducing effectors were identified with differing host subcellular localization patterns, which were named intracellular necrosis‐inducing effector 1–5 (SsINE1–5). We show for the first time a broad host range pathogen effector, SsINE1, that uses an RxLR‐like motif to enter host cells. Furthermore, we provide preliminary evidence that SsINE5 induces necrosis via an NLR protein. All five of the identified effectors are highly conserved in globally sourced S. sclerotiorum isolates. Taken together, these results advance our understanding of the virulence mechanisms employed by S. sclerotiorum and reveal potential avenues for enhancing genetic resistance to this damaging fungal pathogen.

BackgroundPseudomonas fuscovaginae (Pfv) is an emerging plant pathogen of rice and also of other gramineae plants. It causes sheath brown rot disease in rice with symptoms that are characterized by brown lesions on the flag leaf sheath, grain discoloration and sterility. It was first isolated as a high altitude pathogen in Japan and has since been reported in several countries throughout the world. Pfv is a broad host range pathogen and very little is known about its virulence mechanisms.ResultsAn in planta screen of 1000 random independent Tn5 genomic mutants resulted in the isolation of nine mutants which showed altered virulence. Some of these isolates are mutated for functions which are known to be virulence associated factors in other phytopathogenic bacteria (eg. pil gene, phytotoxins and T6SS) and others might represent novel virulence loci.ConclusionsBeing an emerging pathogen worldwide, the broad host range pathogen Pfv has not yet been studied for its virulence functions. The roles of the nine loci identified in the in planta screen are discussed in relation to pathogenicity of Pfv. In summary, this article reports a first study on the virulence of this pathogen involving in planta screening studies and suggests the presence of several virulence features with known and novel functions in the Pseudomonas group of bacteria.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-014-0274-7) contains supplementary material, which is available to authorized users.

Cassava root rot diseases such as dry and black root rot are listed among the major threats, since its affect the main product (tuberous roots), causing a progressive decline in yield and affecting subsequent crop cycles, being the use of resistant varieties the most reliable control measure on field level. The objective of this study was to identify inoculation methods for the early evaluation of genotypes, considering the level of resistance to dry (DRR) and black (BRR) root rot diseases. Different methodologies and plant tissues were evaluated, based on the immersion of cassava tissues (roots and stem cuttings), soil infestation, and inoculation of detached tissues (leaves, tuberous roots and stem cuttings). The following parameters were evaluated for inoculations based on tissue immersion: disease index (ω); aerial part weight (g); fresh weight of the roots (g); and volume (cm3). For the inoculations on detached tissues, the percentage of lesioned area was determined. Immersion methods for roots and lesioned stems did not show typical symptoms of DRR and BRR during the two-month evaluation period. The soil infestation method did not differ from the stem immersion method as to the reduction of aerial part weight and the disease index, whereas both can be recommended for resistance selection trials. There was a positive correlation between the BRR and DRR data for the stem inoculation (r = 0.94, p = 0.001) and for DRR in the peel and root pulp (r = 0.73, p = 0.05). Therefore, the resistance within each tissue is apparently independent and should be compared with the behavior of the genotypes in the field.

Flue-cured tobacco production plays an important role for the most farmers in Yunnan province.However,farmers are not willing to plant Hongda variety,partly because the tobacco seedlings are much susceptible to black shank and black root rot even when fertilized with the same ratio of potassium as for the resistant varieties such as K326 and Yun 85.In addition to the genetic difference in resistance,whether potassium application rate recommended for Hongda variety in the production is sufficient to enhance its resistance to diease is not clear.Therefore,three potassium application rates,including 187.5 (CK,recommended in the production),262.5,and 337.5 kg K2O ha–1,were designed to compare the tobacco seedlings’ resistance to black shank and black root rot in field experiment.The leaves were sampled from healthy tobacco plants (Nicotiana tabacum L.var.Hongda) at 33,41,62,69,76 days after transplanting (DAT),and some physiological-biochemical parameters such as contents of nitrogen,protein,free amino acid,soluble sugar,phenol and flavonoid were determined.Meanwhile,the severity of black shank and black root rot on tobacco plants was recorded in the field at 10-day interval from 24 DAT to 114 DAT.The results showed that the contents of N,protein and free amino acid reduced,the contents of flavonoid,phenol and soluble sugar in leaf increased when potassium application rate was raised.In addition,decreased severity of black shank and black root rot was observed with increasing potassium rate at 44,54,64,74,84,94,104,and 114 DAT.There was significant difference between the two treatments with K2O application rate at 337.5 and 187.5 kg ha–1 respectively in the parameters mentioned above.However,the leaf yield and economic value reached the peak when potassium was applied at 262.5 kg K2O ha–1.Compared with those of the control,the leaf yield and economic value increased by 4.49% and 18.27% respectively in the treatment with potassium application rate at 262.5kg K2O ha–1,while by 1.55% and 12.34% respectively in the treatment with K2O rate at 337.5 kg ha–1.It suggested that potassium application rate recommended in the production of Nicotiana tabacum L.variety Hongda is inadequate,therefore a relatively higher potassium application rate,i.e.262.5 kg K2O ha–1 in the experiment,should be preferred to decrease the severity of black shank and black root rot.

For pathogens infecting single host species evolutionary trade-offs have previously been demonstrated between pathogen-induced mortality rates and transmission rates. It remains unclear, however, how such trade-offs impact sub-lethal pathogen-inflicted damage, and whether these trade-offs even occur in broad host-range pathogens. Here, we examine changes over the past 110 years in symptoms induced in maize by the broad host-range pathogen, maize streak virus (MSV). Specifically, we use the quantified symptom intensities of cloned MSV isolates in differentially resistant maize genotypes to phylogenetically infer ancestral symptom intensities and check for phylogenetic signal associated with these symptom intensities. We show that whereas symptoms reflecting harm to the host have remained constant or decreased, there has been an increase in how extensively MSV colonizes the cells upon which transmission vectors feed. This demonstrates an evolutionary trade-off between amounts of pathogen-inflicted harm and how effectively viruses position themselves within plants to enable onward transmission.

For pathogens infecting single host species evolutionary trade-offs have previously been demonstrated between pathogen-induced mortality rates and transmission rates. It remains unclear, however, how such trade-offs impact sub-lethal pathogen-inflicted damage, and whether these trade-offs even occur in broad host-range pathogens. Here, we examine changes over the past 110 years in symptoms induced in maize by the broad host-range pathogen, maize streak virus (MSV). Specifically, we use the quantified symptom intensities of cloned MSV isolates in differentially resistant maize genotypes to phylogenetically infer ancestral symptom intensities and check for phylogenetic signal associated with these symptom intensities. We show that whereas symptoms reflecting harm to the host have remained constant or decreased, there has been an increase in how extensively MSV colonizes the cells upon which transmission vectors feed. This demonstrates an evolutionary trade-off between amounts of pathogen-inflicted harm and how effectively viruses position themselves within plants to enable onward transmission.

Investigating soilborne nectriaceous fungi impacting avocado tree establishment in Australia

Plant pathogens with a broad host range are commercially more attractive as microbial bioherbicides than strictly host-specific pathogens as a result of the wider market potential of a product capable of controlling multiple species. However, the perceived spatiotemporal disease risk to nontarget plants is a barrier to their adoption for weed control. We consider two approaches to managing this risk. First, we consider safety zones and withholding periods for bioherbicide treatment sites. These must ensure inoculum spreading from, or surviving at the site, exposes nontarget plants to no more inoculum than from natural sources. They can be determined using simple dispersal models. We show that a ratio of added:natural inoculum of 1.0 is biologically reasonable as an 'acceptable risk' and a sound basis for safety zones and withholding periods. These would be analogous to the 'conditions of use' for synthetic chemical herbicides aimed at minimizing collateral damage to susceptible plants from spray drift and persistent soil residues. Second, weed-specific isolates of broad host-range pathogens may avoid the need for safety zones and withholding periods. Such isolates have been found in many broad host-range pathogen species. Their utilization as bioherbicides may more easily meet the requirements of regulators. Mixtures of different weed-specific isolates of a pathogen could provide bioherbicides with commercially attractive spectrums of weed control activity. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Whole-Genome Resource of Lasiodiplodia pseudotheobromae BaA, the Causative Agent of Black Root Rot Morinda officinalis.

Genome sequencing combined with high-throughput functional analyses has proved vital in our quest to understand oomycete-plant interactions. With the identification of effector molecules from Phytophthora spp. we can now embark on dissecting the mechanisms by which effectors modulate host processes and thus ensure parasite fitness. One of the key limitations, however, is to genetically modify Phytophthora and assess gene function during parasitism. Here, we describe a straightforward protocol that allows rapid transformation of Phytophthora capsici, an emerging model in oomycete biology. P. capsici is a broad host range pathogen that can infect a wide variety of plants under lab conditions making it a suitable model for detailed studies on oomycete-host interactions. This protocol relies on electroporation-assisted uptake of DNA in to motile zoospores and allows the rapid identification and characterization of genetically stable transformants.

BackgroundAs an agriculturally important oomycete genus, Phytophthora contains a large number of destructive plant pathogens that severely threaten agricultural production and natural ecosystems. Among them is the broad host range pathogen P. palmivora, which infects many economically important plant species. An essential way to dissect their pathogenesis mechanisms is genetic modification of candidate genes, which requires effective transformation systems. Four methods were developed for transformation of Phytophthora spp., including PEG(polyethylene glycol)/CaCl2 mediated protoplast transformation, electroporation of zoospores, microprojectile bombardment and Agrobacterium-mediated transformation (AMT). Among them, AMT has many advantages over the other methods such as easy handling and mainly generating single-copy integration in the genome. An AMT method previously reported for P. infestans and P. palmivora has barely been used in oomycete research due to low success and low reproducibility.ResultsIn this study, we report a simple and efficient AMT system for P. palmivora. Using this system, we were able to reproducibly generate over 40 transformants using zoospores collected from culture grown in a single 100 mm-diameter petri dish. The generated GFP transformants constitutively expressed GFP readily detectable using a fluorescence microscope. All of the transformants tested using Southern blot analysis contained a single-copy T-DNA insertion.ConclusionsThis system is highly effective and reproducible for transformation of P. palmivora and expected to be adaptable for transformation of additional Phytophthora spp. and other oomycetes. Its establishment will greatly accelerate their functional genomic studies.

Recent research has shown that cyclophilins, proteins that catalyze the isomerization of peptidyl-prolyl bonds, play a variety of important roles in infection, including facilitating host penetration and colonization and activating pathogen effector proteins within the host cytoplasm. In the current study, bioinformatic analysis of the genomes of three species of plant pathogens in the genus Phytophthora has revealed extensive synteny between the 20 or 21 members of the cyclophilin gene family. In P. infestans, extensive EST studies give evidence of the expression of 14 of the 21 genes. Sequences homologous to 12 of the 14 expressed P. infestans cyclophilins were isolated using PCR and gene-specific primers in the broad host range pathogen, P. nicotianae. Quantitative real-time PCR measurements of transcript levels in P. nicotianae at four stages of asexual development and during infection of resistant and susceptible tobacco plants gave evidence of expression of seven of the P. nicotianae homologs. The most abundantly expressed gene, PnCyPA, has a lower mRNA level in zoospores compared to other stages of asexual development and its expression increases during infection of susceptible plants. Immunocytochemical studies indicate that PnCyPA occurs in the nucleus and cytoplasm of P. nicotianae cells and is secreted from germinated cysts.

Oomycetes, of the genus Phytophthora, comprise of some of the most devastating plant pathogens. Parasitism of Phytophthora results from evolution from an autotrophic ancestor and adaptation to a wide range of environments, involving metabolic adaptation. Sequence mining showed that Phytophthora spp. display an unusual repertoire of glycolytic enzymes, made of multigene families and enzyme replacements. To investigate the impact of these gene duplications on the biology of Phytophthora and, eventually, identify novel functions associated to gene expansion, we focused our study on the first glycolytic step on P. nicotianae, a broad host range pathogen. We reveal that this step is committed by a set of three glucokinase types that differ by their structure, enzymatic properties, and evolutionary histories. In addition, they are expressed differentially during the P. nicotianae life cycle, including plant infection. Last, we show that there is a strong association between the expression of a glucokinase member in planta and extent of plant infection. Together, these results suggest that metabolic adaptation is a component of the processes underlying evolution of parasitism in Phytophthora, which may possibly involve the neofunctionalization of metabolic enzymes.