Identification and florfenicol-treatment of pseudomonas putida infection in gilthead seabream (Sparus aurata) fed on tilapia-trash-feed.

The present study aimed to determine the major cause of the high mortality affecting farmed gilthead seabream (Sparus aurata) and controlling this disease condition. Fifteen diseased S. aurata were sampled from a private fish farm located at Eldeba Triangle, Damietta, fish showed external skin hemorrhages, and ulceration. Bacterial isolates retrieved from the diseased fish were identified biochemically as Pseudomonas putida and then confirmed by phylogenetic analysis of the 16 S rRNA gene sequence. P. putida was also isolated from three batches of tilapia-trash feed given to S. aurata. Biofilm and hemolytic assay indicated that all P. putida isolates produced biofilm, but 61.11% can haemolyse red blood cells. Based on the antibiotic susceptibility test results, P. putida was sensitive to florfenicol with minimum inhibitory concentrations ranging between 0.25 and 1.0 µg mL- 1, but all isolates were resistant to ampicillin and sulfamethoxazole-trimethoprim. Pathogenicity test revealed that P. putida isolate (recovered from the tilapia-trash feed) was virulent for S. aurata with LD50 equal to 4.67 × 107 colony forming unit (CFU) fish- 1. After intraperitoneal (IP) challenge, fish treated with 10 mg kg- 1 of florfenicol showed 16.7% mortality, while no mortality was recorded for the fish group that received 20 mg kg- 1. The non-treated fish group showed 46.7% mortality after bacterial challenge. HPLC analysis of serum florfenicol levels reached 1.07 and 2.52 µg mL- 1 at the 5th -day post-drug administration in the fish groups received 10 and 20 mg kg- 1, respectively. In conclusion, P. putida was responsible for the high mortality affecting cultured S. aurata, in-feed administration of florfenicol (20 mg kg- 1) effectively protected the challenged fish.

Biological characteristics and pathogenicity comparison of Nocardia seriolae isolated from Micropterus salmoides and Channa argus.

Nocardia seriolae is the primary pathogen causing nocardiosis in various fish species, leads to significant economic losses in the aquaculture industry. In this study, 10 bacterial strains isolated from Micropterus salmoides and Channa argus infected with nocardiosis, were identified as N. seriolae by physiological and biochemical identification, as well as 16S rDNA sequencing. Moreover, the key virulence-related genes such as ESX-1, T7SS-2, T7SS-3, EspG1, sodC, sod2 and ESAT6 were all positive, and showing high homology among different strains. Pathogenicity testing revealed mortality rates ranging from 70 to 100%, accompanied by the presence of white nodules in the viscera of deceased fish. The drug sensitivity test demonstrated that LY21811, the most lethal strain, exhibited high sensitivity to nine types of antibiotics, including azithromycin, doxycycline, florfenicol and compound sulfamethoxazole, yet showed complete resistance to ß-lactam antibiotics. Additionally, the tannic acid also demonstrated potent inhibitory effects against LY21811, with a minimum inhibitory concentration of 0.0625 mg/mL. These results showed that N. seriolae originated from M. salmoides and C. argus in Zhejiang Province were highly conserved, demonstrating a high homogeneity in genetic characteristics, pathogenicity and antimicrobial susceptibilities. These results provide a foundation for further research on the pathogenic characteristics and disease prevention of N. seriolae infections.

Rhizopus stolonifer and related control strategies in postharvest fruit: A review.

Rhizopus stolonifer is one of the main pathogens in postharvest storage logistics of more than 100 kinds of fruit, such as strawberries, tomatoes and melons. In this paper, the research on the morphology and detection, pathogenicity and infection mechanism of Rhizopus stolonifer was reviewed. The control methods of Rhizopus stolonifer in recent years was summarized from three dimensions of physics, chemistry and biology, including the nanomaterials, biological metabolites, light control bacteria, etc. Future direction of postharvest Rhizopus stolonifer infection control was analyzed from two aspects of pathogenic mechanism research and new composite technology. The information provided in this review will help researchers and technicians to deepen their understanding of the pathogenicity of Rhizopus stolonifer, and develop more effective control methods in the future.

Antimicrobial resistance of Pseudomonas aeruginosa: navigating clinical impacts, current resistance trends, and innovations in breaking therapies.

Pseudomonas aeruginosa, a Gram-negative bacterium, is recognized for its adaptability and opportunistic nature. It poses a substantial challenge in clinical settings due to its complicated antibiotic resistance mechanisms, biofilm formation, and capacity for persistent infections in both animal and human hosts. Recent studies revealed a potential zoonotic transmission of P. aeruginosa between animals, the environment, and human populations which highlights awareness of this microbe. Implementation of the One Health approach, which underscores the connection between human, animal, and environmental health, we aim to offer a comprehensive perspective on the current landscape of P. aeruginosa management. This review presents innovative strategies designed to counteract P. aeruginosa infections. Traditional antibiotics, while effective in many cases, are increasingly compromised by the development of multidrug-resistant strains. Non-antibiotic avenues, such as quorum sensing inhibition, phage therapy, and nanoparticle-based treatments, are emerging as promising alternatives. However, their clinical application encounters obstacles like cost, side effects, and safety concerns. Effectively addressing P. aeruginosa infections necessitates persistent research efforts, advancements in clinical development, and a comprehension of host-pathogen interactions to deal with this resilient pathogen.

Laboratory Evaluation of Indigenous and Commercial Entomopathogenic Nematodes against Red Palm Weevil, Rhynchophorus ferrugineus (Coleoptera: Curculionidae).

The red palm weevil (RPW) is a significant threat to date palms. Conventional pest control has been ineffective. This study aims to evaluate entomopathogenic nematodes (EPNs) indigenous to Saudi Arabia and commercial against RPW. We used 33 soil samples collected from four areas of Saudi Arabia. The indigenous EPNs were isolated and cultured using an insect baiting method to obtain infective juveniles. Pathogenicity bioassays were conducted against different stages of RPW, including eggs, larvae, and adults. The bioassay was performed using all the isolates at 1 × 106 IJ/mL. Distilled water was used as a control. The results revealed that only 9.09% of soil samples contained positive EPNs. Through DNA sequencing analysis, the positive samples were identified as indigenous isolates belonging to Heterorhabditis indica and Steinernema carpocapsae EPN species. In pathogenicity tests, 90% mortality of RPW eggs was observed after five days. Similar mortality trends were seen in RPW larvae and adults, with 90% mortality recorded after ten days for all the EPN treatments. Mortality increased with the duration of post-EPN inoculation exposure. The 1 × 106 IJ/mL concentrations of EPN effectively killed various stages of RPW in the laboratory. More research is needed to test EPNs against RPW in the field.

Discovery and Transcriptional Profiling of Penicillium digitatum Genes That Could Promote Fungal Virulence during Citrus Fruit Infection.

Green mold caused by Penicillium digitatum (Pers.:Fr.) Sacc is the most prevalent postharvest rot concerning citrus fruits. Using the subtractive suppression hybridization (SSH) technique, different P. digitatum genes have been identified that could be involved in virulence during citrus infection in the early stages, a crucial moment that determines whether the infection progresses or not. To this end, a comparison of two P. digitatum strains with high and low virulence has been carried out. We conducted a study on the gene expression profile of the most relevant genes. The results indicate the importance of transcription and regulation processes as well as enzymes involved in the degradation of the plant cell wall. The most represented expressed sequence tag (EST) was identified as PDIP_11000, associated with the FluG domain, which is putatively involved in the activation of conidiation. It is also worth noting that PDIP_02280 encodes a pectin methyl esterase, a cell wall remodeling protein with a high expression level in the most virulent fungal strains, which is notably induced during citrus infection. Furthermore, within the group with the greatest representation and showing significant induction in the early stages of infection, regulatory proteins (PDIP_68700, PDIP_76160) and a chaperone (PDIP_38040) stand out. To a lesser extent, but not less relevant, it is worth distinguishing different regulatory proteins and transcription factors, such as PDIP_00580, PDIP_49640 and PDIP_78930.

The APSES Transcription Factor SsStuA Regulating Cell Wall Integrity Is Essential for Sclerotia Formation and Pathogenicity in Sclerotinia sclerotiorum.

APSES (Asm1p, Phd1p, Sok2p, Efg1p, and StuAp) family transcription factors play crucial roles in various biological processes of fungi, however, their functional characterization in phytopathogenic fungi is limited. In this study, we explored the role of SsStuA, a typical APSES transcription factor, in the regulation of cell wall integrity (CWI), sclerotia formation and pathogenicity of Sclerotinia sclerotiorum, which is a globally important plant pathogenic fungus. A deficiency of SsStuA led to abnormal phosphorylation level of SsSmk3, the key gene SsAGM1 for UDP-GlcNAc synthesis was unable to respond to cell wall stress, and decreased tolerance to tebuconazole. In addition, ΔSsStuA was unable to form sclerotia but produced more compound appressoria. Nevertheless, the virulence of ΔSsStuA was significantly reduced due to the deficiency of the invasive hyphal growth and increased susceptibility to hydrogen peroxide. We also revealed that SsStuA could bind to the promoter of catalase family genes which regulate the expression of catalase genes. Furthermore, the level of reactive oxygen species (ROS) accumulation was found to be increased in ΔSsStuA. In summary, SsStuA, as a core transcription factor involved in the CWI pathway and ROS response, is required for vegetative growth, sclerotia formation, fungicide tolerance and the full virulence of S. sclerotiorum.

Characterizing the Palm Pathogenic Thielaviopsis Species from Florida.

Thielaviopsis paradoxa sensu lato is a soilborne fungal pathogen that causes Thielaviopsis trunk rot and heart rot in palms. The loss of structural integrity resulting from trunk rot can cause the palm trunk to collapse suddenly and poses a serious threat to life and property. Even though rudimentary knowledge about the Thielaviopsis infection process in palms is available, nothing is known about the T. paradoxa species complex in the US. The aim of this study was to characterize T. paradoxa s. lat. isolates collected from diseased palms grown in Florida. Multi-locus phylogeny using three genes, ITS, ß-tubulin, and tef1-α, revealed that the isolates separate into two distinct clades with high bootstrap support. The majority of the isolates clustered with the species T. ethacetica, while two isolates formed a separate clade, distinct from T. musarum, and might represent an undescribed Thielaviopsis species. One representative isolate from each clade, when grown on three distinct media and at four different temperatures, showed differences in gross colony morphology, as well as growth rates. The T. ethacetica isolate TP5448 and the Thielaviopsis sp. isolate PLM300 grew better at opposite ends of the temperature spectrum tested in this study, i.e., 35 °C and 10 °C, respectively. In pathogenicity assays on whole plants, the T. ethacetica isolate proved to be more aggressive than Thielaviopsis sp. isolate PLM300, as it produced larger lesions when inoculated on wounded leaflets. An unequal distribution was observed for the mating-type locus of T. ethacetica, as 12 isolates carried the MAT1-1-1 allele, while the status for four isolates remained undefined. Variation in mycelial growth in response to different fungicides was also observed between the two clades. These results demonstrate the existence of two Thielaviopsis clades that can infect palms in Florida and underscore the need for targeted sampling to help uncover the diversity of Thielaviopsis species across palm-growing regions in the US.

Histone Methyltransferase SsDim5 Regulates Fungal Virulence through H3K9 Trimethylation in Sclerotinia sclerotiorum.

Histone post-translational modification is one of the main mechanisms of epigenetic regulation, which plays a crucial role in the control of gene expression and various biological processes. However, whether or not it affects fungal virulence in Sclerotinia sclerotiorum is not clear. In this study, we identified and cloned the histone methyltransferase Defective in methylation 5 (Dim5) in S. sclerotiorum, which encodes a protein containing a typical SET domain. SsDim5 was found to be dynamically expressed during infection. Knockout experiment demonstrated that deletion of SsDim5 reduced the virulence in Ssdim5-1/Ssdim5-2 mutant strains, accompanied by a significant decrease in H3K9 trimethylation levels. Transcriptomic analysis further revealed the downregulation of genes associated with mycotoxins biosynthesis in SsDim5 deletion mutants. Additionally, the absence of SsDim5 affected the fungus's response to oxidative and osmotic, as well as cellular integrity. Together, our results indicate that the H3K9 methyltransferase SsDim5 is essential for H3K9 trimethylation, regulating fungal virulence throug mycotoxins biosynthesis, and the response to environmental stresses in S. sclerotiorum.

FgFAD12 Regulates Vegetative Growth, Pathogenicity and Linoleic Acid Biosynthesis in Fusarium graminearum.

Polyunsaturated fatty acids (PUFAs), as important components of lipids, play indispensable roles in the development of all organisms. ∆12 fatty acid desaturase (FAD12) is a speed-determining step in the biosynthesis of PUFAs. Here, we report the characterization of FAD12 in Fusarium graminearum, which is the prevalent agent of Fusarium head blight, a destructive plant disease worldwide. The results demonstrated that deletion of the FgFAD12 gene resulted in defects in vegetative growth, conidial germination and plant pathogenesis but not sexual reproduction. A fatty acid analysis further proved that the deletion of FgFAD12 restrained the reaction of oleic acid to linoleic acid, and a large amount of oleic acid was detected in the cells. Moreover, the ∆Fgfad12 mutant showed increased resistance to osmotic stress and reduced tolerance to oxidative stress. The expression of FgFAD12 did show a temperature-dependent manner, which was not affected at a low temperature of 10 °C when compared to 25 °C. RNA-seq analysis further demonstrated that most genes enriched in fatty acid metabolism, the biosynthesis of unsaturated fatty acids, fatty acid biosynthesis, fatty acid degradation, steroid biosynthesis and fatty acid elongation pathways were significantly up-regulated in the ∆Fgfad12 mutants. Overall, our results indicate that FgFAD12 is essential for linoleic acid biosynthesis and plays an important role in the infection process of F. graminearum.

The Comparative Genomics of Botryosphaeriaceae Suggests Gene Families of Botryosphaeria dothidea Related to Pathogenicity on Chinese Hickory Tree.

Trunk canker poses a major threat to the production of Chinese hickory tree (Carya cathayensis Sarg.), which is primarily determined by Botryosphaeriaceae. In our previous work, we identified Botryosphaeria dothidea as the predominant pathogen of this disease. However, it is still unclear about corresponding gene families and mechanisms associated with B. dothidea's pathogenicity on Chinese hickory tree. Here, we present a comparative analysis of high-quality genome assemblies of Botryosphaeria dothidea and other isolated pathogens, showing highly syntenic relationships between B. dothidea and its closely related species and the conservative evolution of the Botryosphaeriaceae family. Higher GC contents were found in the genomes of B. dothidea and three other isolated pathogens (Botryshaeria cortices, Botryshaeria fabicerciana, and Botryshaeria qingyuanensis) compared to Macrophomina phaseolina, Neofusicoccum parvum, Diplodia corticola, and Lasiodiplodia theobromae. An investigation of genes specific to or expanded in B. dothidea revealed that one secreted glucanase, one orsellinic acid biosynthesis enzyme, and two MFS transporters positively regulated B. dothidea's pathogenicity. We also observed an overrepresentation of viral integrase like gene and heterokaryon incompatibility proteins in the B. dothidea's genome. In addition, we observed one LRR-domain-containing protein and two Sec-domain-containing proteins (Sec_1 and Sec_7) that underwent positive selection. This study will help to understand B. dothidea's pathogenicity and potential influence on the infection of Chinese hickory, which will help in the development of disease control and ensure the security of Chinese hickory production.

Pathotyping Systems and Pathotypes of Plasmodiophora brassicae-Navigating toward the Optimal Classification.

Plasmodiophora brassicae Woronin, an obligate biotrophic soil-borne pathogen, poses a significant threat to cruciferous crops worldwide by causing the devastating disease known as clubroot. Pathogenic variability in P. brassicae populations has been recognized since the 1930s based on its interactions with Brassica species. Over time, numerous sets of differential hosts have been developed and used worldwide to explore the pathogenic variability within P. brassicae populations. These sets encompass a range of systems, including the Williams system, the European Clubroot Differential set (ECD), the Brassica napus set, the Japanese Clubroot Differential Set, the Canadian Clubroot Differential Set (CCS), the Korean Clubroot Differential Set, and the Chinese Sinitic Clubroot Differential set (SCD). However, all existing systems possess both advantages as well as limitations regarding the detection of pathotypes from various Brassica species and their corresponding virulence pattern on Brassica genotypes. This comprehensive review aims to compare the main differential systems utilized in classifying P. brassicae pathotypes worldwide. Their strengths, limitations, and implications are evaluated, thereby enhancing our understanding of pathogenic variability.

The Gene vepN Regulated by Global Regulatory Factor veA That Affects Aflatoxin Production, Morphological Development and Pathogenicity in Aspergillus flavus.

Velvet (VeA), a light-regulated protein that shuttles between the cytoplasm and the nucleus, serves as a key global regulator of secondary metabolism in various Aspergillus species and plays a pivotal role in controlling multiple developmental processes. The gene vepN was chosen for further investigation through CHIP-seq analysis due to significant alterations in its interaction with VeA under varying conditions. This gene (AFLA_006970) contains a Septin-type guanine nucleotide-binding (G) domain, which has not been previously reported in Aspergillus flavus (A. flavus). The functional role of vepN in A. flavus was elucidated through the creation of a gene knockout mutant and a gene overexpression strain using a well-established dual-crossover recombinational technique. A comparison between the wild type (WT) and the ΔvepN mutant revealed distinct differences in morphology, reproductive capacity, colonization efficiency, and aflatoxin production. The mutant displayed reduced growth rate; dispersion of conidial heads; impaired cell wall integrity; and decreased sclerotia formation, colonization capacity, and aflatoxin levels. Notably, ΔvepN exhibited complete growth inhibition under specific stress conditions, highlighting the essential role of vepN in A. flavus. This study provides evidence that vepN positively influences aflatoxin production, morphological development, and pathogenicity in A. flavus.

Isolation and genetic characterization of multiple genotypes of both H5 and H7 avian influenza viruses from environmental water in the Izumi plain, Kagoshima prefecture, Japan during the 2021/22 winter season.

In the 2021/22 winter, one H5N1 and nine H5N8 high pathogenicity avian influenza viruses (HPAIVs) of clade 2.3.3.4b were isolated from the water in crane roosts on the Izumi plain, Japan. Additionally, we isolated low pathogenicity avian influenza viruses (LPAIVs) of five subtypes: H1N1, H4N2, H4N6, H7N7, and H10N4. H5N8 HPAIVs belonging to the G2a group were isolated throughout winter, whereas H5N1 HPAIV belonging to the G2b group were isolated only in early winter. These findings suggest co-circulation of both G2a and G2b HPAIVs in early winter. Although two H7N7 LPAIVs were isolated from cranes' roost water collected on the same day, the gene constellations of the two isolates were clearly different, indicating the contemporary invasion of at least two different genotypes of H7N7 LPAIVs in the Izumi plain. This study underscores the importance of monitoring both HPAIVs and LPAIVs to understand avian influenza virus ecology in migratory waterfowl populations.

Evolution of H7N9 highly pathogenic avian influenza virus in the context of vaccination.

Human infections with the H7N9 influenza virus have been eliminated in China through vaccination of poultry; however, the H7N9 virus has not yet been eradicated from poultry. Carefully analysis of H7N9 viruses in poultry that have sub-optimal immunity may provide a unique opportunity to witness the evolution of highly pathogenic avian influenza virus in the context of vaccination. Between January 2020 and June 2023, we isolated 16 H7N9 viruses from samples we collected during surveillance and samples that were sent to us for disease diagnosis. Genetic analysis indicated that these viruses belonged to a single genotype previously detected in poultry. Antigenic analysis indicated that 12 of the 16 viruses were antigenically close to the H7-Re4 vaccine virus that has been used since January 2022, and the other four viruses showed reduced reactivity with the vaccine. Animal studies indicated that all 16 viruses were nonlethal in mice, and four of six viruses showed reduced virulence in chickens upon intranasally inoculation. Importantly, the H7N9 viruses detected in this study exclusively bound to the avian-type receptors, having lost the capacity to bind to human-type receptors. Our study shows that vaccination slows the evolution of H7N9 virus by preventing its reassortment with other viruses and eliminates a harmful characteristic of H7N9 virus, namely its ability to bind to human-type receptors.

Effects of TrPLDs on the pathogenicity of Trichothecium roseum infected apple fruit.

Phospholipase D plays a critical regulatory role in the pathogenicity of filamentous fungi. However, the molecular mechanism of PLD regulating the pathogenicity of filamentous fungi has not been reported. In this research, the previously constructed TrPLD1 and TrPLD2 (TrPLDs) mutants were used as test strains. Firstly, the function of TrPLDs in Trichothecium roseum was studied. Then, the effects of TrPLDs on the pathogenicity of T. roseum and the quality of the inoculated apples were verified. The results suggested that the deletion of TrPLD1 delayed the spore germination of ΔTrPLD1 and inhibited germ tube elongation by down-regulating the expressions of TrbrlA, TrabaA and TrwetA. By down-regulating the extracellular enzyme-coding gene expressions, ΔTrPLD1 inhibited the degradation of apple fruit cell wall and the change of fatty acid content during infection, reduced the cell membrane permeability and malondialdehyde (MDA) content of apple fruit, thereby maintaining the integrity of fruit cell membrane, and reduced the pathogenicity of ΔTrPLD1 to apple and kept the quality of apple. However, ΔTrPLD2 did not have a significant effect on the infection process of apple fruit by the pathogen.

Benzoxazinoids secreted by wheat root weaken the pathogenicity of Fusarium oxysporum f. sp. fabae by inhibiting linoleic acid and nucleotide metabolisms.

KEY MESSAGE: The regulatory action of BXs secreted by wheat on the pathogenicity of FOF causing Fusarium wilt in faba bean were analyzed. DIMBOA and MBOA weakened the pathogenicity of FOF. A large number of pathogenic bacteria in continuous cropping soil infect faba bean plants, leading to the occurrence of wilt disease, which restricts their production. Faba bean-wheat intercropping is often used to alleviate this disease. This study investigates the effect of benzoxazinoids (BXs) secreted by wheat root on the pathogenicity of Fusarium oxysporum f. sp. Fabae (FOF) and underlying molecular mechanisms. The effects of DIMBOA(2,4-dihydroxy-7-methoxy-1,4-benzoxazine-4-one) and MBOA(6-methoxybenzoxazolin-2-one) on the activity of cell-wall-degrading enzymes in FOF(cellulase, pectinase, amylase, and protease), FOF Toxin (fusaric acid, FA) content were investigated through indoor culture experiments. The effect of BXs on the metabolic level of FOF was analyzed by metabonomics to explore the ecological function of benzoxazines intercropping control of Fusarium wilt in faba bean. The results show that the Exogenous addition of DIMBOA and MBOA decreased the activity of plant-cell-wall-degrading enzymes and fusaric acid content and significantly weakened the pathogenicity of FOF. DIMBOA and MBOA significantly inhibited the pathogenicity of FOF, and metabolome analysis showed that DIMBOA and MBOA reduced the pathogenicity of FOF by down-regulating related pathways such as nucleotide metabolism and linoleic acid metabolism, thus effectively controlling the occurrence of Fusarium wilt in faba bean.

Compound heterozygous mutations in three Chinese patients of Segawa syndrome and their treatment outcomes.

Segawa syndrome is a rare autosomal recessive form of dopa-responsive dystonia resulting from TH gene dysfunction. Patients typically exhibit symptoms such as generalized dystonia, rigidity, tremors, infantile Parkinsonism, and pseudo-spastic paraplegia. Levodopa is often an effective treatment. Due to its rarity, high heterogeneity, and poorly understood pathological mutation and phenotype spectrums, as well as genotype-phenotype and genotype-treatment outcome correlations, Segawa syndrome poses diagnostic and therapeutic challenges. In our study, through clinical and molecular analyses of three Chinese Segawa patients, we re-evaluated the pathogenicity of a TH mutation (c.880G>C;p.G294R) previously categorized as "Conflicting classifications of pathogenicity" in ClinVar. Also, we summarized the clinical phenotypes of all reported Segawa syndrome cases until 2023 and compared them with our patients. We identified a novel phenotype, "cafe-au-lait macules," not previously observed in Segawa patients. Additionally, we discussed the correlation between specific genotypes and phenotypes, as well as genotypes and treatment outcomes of our three cases. Our findings aim to enhance the understanding of Segawa syndrome, contributing to improved diagnosis and treatment approaches in the future.

First Report of Tobacco Leaf Tip Blight Caused by Alternaria gossypina in China.

Tobacco (Nicotiana tabacum L.) belongs to the family Solanaceae, an economically significant crop (Zhou et al. 2023). Twelve samples with leaf spots were collected in Keti Village, Changshun County, Zunyi City, Guizhou province, China in 2022. Twenty-five percent of the samples had dry lesions near the leaf tip which resulted leaf tip blight after development. Fungi were isolated by a previous method (Wei et al. 2022). Six Alternaria strains were obtained and preserved in the Fungal Herbarium of Yangtze University (YZU), Jingzhou, Hubei, China. Among them, one strain YZU 221477 showed distinct cultural characteristics out of five A. alternata strains, which was again determined by growing on potato dextrose agar (PDA) at 25°C for 7 days in dark to evaluate. The colonies (60 mm in diameter) were white cottony in the center surrounded by vinaceous purple. To examine the morphology, mycelia were inoculated onto potato carrot agar (PCA) at 22°C, following an 8 h light/16 h dark photoperiod (Simmons 2007). Conidia were obclavate or ovoid, normally 3-5 conidial units per chain, 20-38 × 10-16.5 µm, 3 to 5 transverse septa, beakless or a short beak (4-30 µm). The observation results were consistent with those of A. gossypina (Zhang 2003). Total genomic DNA was extracted using the CTAB method and seven gene regions including internal transcribed spacer of rDNA (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1 alpha (TEF1), RNA polymerase second largest subunit (RPB2), Alternaria major allergen gene (Alt a 1), endopolygalacturonase (EndoPG) and an anonymous gene region (OPA10-2) were amplified with ITS5/ITS4, gpd1/gpd2, EF1-728F/EF1-986R, RPB2-5F/RPB2-7cR, Alt-for/Alt-rev, PG3/PG2b and OPA10-2L/OPA10-2R primers, respectively. All sequences were deposited in GenBank (ITS: OR710806; GAPDH: PP057862; TEF1: PP158601; RPB2: PP057863; Alt a 1: PP057865; EndoPG: PP057861; OPA10-2: PP057864). Combining with relevant sequences retrieved from the NCBI database were used for the phylogenetic analysis. Maximum Likelihood (ML) tree was constructed with RAxML v.7.2.8 employing GTRCAT model using 1000 bootstrap (BS) replicates to assess statistical support. The results indicated that the present strain grouped with A. gossypina (type strain of CBS 104.32) supported with 73% bootstrap values, also having a support of 0.83 Bayesian posterior probabilities values. Based on morphology and molecular evidence, the strain YZU 221477 is identified as Alternaria gossypina. Pathogenicity was examined to fulfill Koch's postulates. Mycelial plugs (6 mm diameter) of the present strain and A. alternata cultivated on PDA were taken from the margin and inoculated onto viable tobacco leaves (Cultivar: Yunyan 87, n=3) growing forty days, while controls were inoculated with sterile PDA. The assay was conducted three times. The plants were maintained at 25°C with humidity levels over 85% in a greenhouse. Leaves were evaluated after 7 days, necrotic spots encircled by yellow halos were on both inoculums, except controls. Pathogen re-isolation confirmed that it was the same as inoculated fungus based on morphology. A. gossypina was firstly found on cotton (Hopkins 1931), late reported to induce disease on Minneola, Nopalea, Hibiscus, Citrus, Solanum and Ageratina. To our knowledge, this is the first report of A. gossypina causing tobacco leaf tip blight in China, and it also provides a basis for controlling of tobacco leaf tip blight.

Molecular assessment of oat head blight fungus, including a new genus and species in a family of Nectriaceae.

Head blight (HB) of oat (Avena sativa) has caused significant production losses in oats growing areas of western China. A total of 314 isolates, associated with HB were collected from the major oat cultivating areas of Gansu, Qinghai, and Yunnan Provinces in western China. Based on morphological characters, the isolates were initially classified into three genera, as differentiation to species was a bit difficult. Taxonomic analysis of these isolates based on muti-gene phylogenetic analyses (ITS, TEF1, TUB2, and RPB2) revealed four known Fusarium species, F. proliferatum, F. avenaceum, F. poae, and F. sibiricum, and one Acremonium specie (A. sclerotigenum). In addition, a new genus Neonalanthamala gen. nov., similar to genus Nalanthamala was introduced herein with a new combination, Neonalanthamala graminearum sp. nov., to accommodate the HB fungus. The molecular clock analyses estimated the divergence time of the Neonalanthamala and Nalanthamala based on a dataset (ITS, TUB2, RPB2), and we recognized the mean stem ages of the two genera are 98.95 Mya, which showed that they evolved from the same ancestor. N. graminearum was the most prevalent throughout the surveyed provinces. Pathogenicity test was carried out by using two different methods: seed inoculation and head inoculation. Results showed that F. sibiricum isolates were the most aggressive on the seed and head. A. sclerotigenum isolates were not pathogenic to seeds, and were developed less symptoms to the head compared to other species. Data analyses showed that the correlation of the germination potential, germination index, and dry weight of seed inoculation and disease index of plant inoculation had a highly significant negative correlation (P < 0.001). These results showed that the development of HB might be predicted by seed tests for this species. A. sclerotigenum and N. graminearum causing HB are being firstly reported on oat in the world. Similarly, F. proliferatum, F. avenaceum, F. poae and F. sibiricum causing oat HB are firstly reported in China.