Translation Elongation Factor 1α Gene Research Articles

Molecular Identification and Antifungal Susceptibility of Fusarium spp. Clinical Isolates.

Accurate identification of Fusarium species requires molecular identification. Treating fusariosis is challenging due to widespread antifungal resistance, high rates of treatment failure, and insufficient information relating antifungal susceptibility to the clinical outcome. Despite recent outbreaks in Mexico, there is limited information on epidemiology and antifungal susceptibility testing (AST). We aimed to analyse the distribution of Fusarium species from a referral centre in Mexico with DNA sequencing and to describe AST to the clinical outcome. We conducted a retrospective study on clinical isolates of Fusarium. They were identified by translation elongation factor-1α gene amplification and sequencing. AST was performed to determine minimal inhibitory concentrations (MICs). A total of 35 Fusarium isolates from 26 patients were included. The most common was Fusarium solani species complex (FSSC) in 51.5%, of which Fusarium petroliphilum and Fusarium oxysporum species complex were the most frequent with 37% and 20%, respectively. AST did not show MICs above the epidemiological cut-off value. Fusariosis was diagnosed in 19 patients, mostly with hematologic neoplasm; the overall mortality rate was 32%. Fusarium petroliphilum from the FSSC was found most frequently. Elevated mortality and MICs for all tested antifungals were found, with higher MIC50 among F. solani SC than F. oxysporum SC or F. fujikuroi SC.

First report of leaf brown spot caused by Diaporthe phoenicicola on Lithocarpus polystachyus in China.

The leaves of Lithocarpus polystachyus (Wall. ex A. DC.), an economically significant tree species in China, are commonly referred to as 'sweat tea' due to their high dihydrochalcone content, which holds biomedical importance, particularly in the treatment of diabetes (Hou et al. 2011). In January 2024, brown spots on L. polystachyus leaves were widely observed in Ningxiang (28°23'N, 112°59'E), Hunan Province, China. According to the investigation, the incidence rate of this disease was about 74% (222/300 plants surveyed). On each infected plant, nearly 60% leaves had symptoms. The disease initially presented as small yellow lesions that eventually developed into large brown patches with dark brown edges. More than 80% of the area was covered by leaf lesions, which eventually turned into leaf necrosis. To ascertain the pathogenic species responsible for this disease, pathogen isolation was conducted using a tissue separation method (Xu et al. 2023). The infected leaf tissues were surface-disinfected by immersing in 75% ethanol followed by 0.1% HgCl2. Small pieces (0.5 × 0.5 cm) were then excised and placed onto PDA medium, and incubated at 28°C for 6-9 days. Sterilized dissecting needles were used to pick mycelia from the edge of the colonies and placed onto PDA for strains purification. On the PDA, the colony color of upper side initially appeared white (Rayner 1A1), and then turned grey (Rayner 11C1), while the reverse side turnd faint yellow (Rayner 4A3). Black pycnidia were induced on PDA at 28°C under a 12 h/12 h light/dark cycle for 12 days. Alpha conidia were 5.37 _8.84 × 1.53 _3.19 μm (average: 6.77 × 2.37 μm, n = 50), hyaline, fusiform or ellipsoidal. Beta conidia were 13.61 _23.45 μm × 0.94 _1.47 μm (average: 18.78×1.18 μm, n = 50), hyaline, aseptate, filiform, straight or hamate. Morphologically, the fungi were identified as Diaporthe sp. (Guarnaccia and Crous 2017). To further affirm the identification of the pathogen, the genomic DNA was extracted from representative isolate, referred to as Dip, for molecular identification. The internal transcribed spacer region (ITS), translation elongation factor 1α (EF1-α), β-tubulin (TUB2) and histone H3 (HIS) genes were amplified from genomic DNA using primers ITS1/ITS4, EF1-728F/EF1-986R, Bt2a/Bt2b, and CYLH3F/H3-1b, respectively, to sequence for BLAST (Huang et al. 2015). The results showed that the ITS (GenBank: PP502145), EF1-α (GenBank: PP505773), TUB2 (GenBank: PP505774) and HIS (GenBank: PP505772) sequences of Dip isolate, respectively, showed 100% (469/469 bp), 98.47% (257/261 bp), 97.63% (617/632 bp), and 100% (379/379 bp) identity to their counterparts (GenBank: MW504747, ON049530, MW514138 and ON113058) in Diaporthe phoenicicola. The Maximum Likelihood tree was built based on the ITS, EF1-α, HIS and TUB2 sequences using MEGA11.0. Isolate Dip clustered with D. phoenicicola. The fungus was finally identified as D. phoenicicola by combining morphological and molecular characteristics. To confirm the pathogenicity of the isolated D. phoenicicola to induce brown spot, the pathogenicity assay was assessed following Koch's postulates (Gradmann, 2014). Conidial suspension (1×105 conidia per mL) was inoculated on 12 unwounded leaves collected from six 3-years-old plants, and sterile water was as control. The inoculated leaves were then incubated in chambers at 28℃ and 90% humidity with a 12 h photoperiod. The experiment was repeated three times. The results showed that inoculated leaves other than control developed brown spot symptoms within six days after inoculation. The test proved D. phoeicicola as the causal agent of this brown spot disease on L. polystachyus. The pathogen was exclusively re-isolated from the infected leaves and showed identical morphological characteristics to those of the original pathogens. To our knowledge, this is the first report of leaf brown spot of L. polystachyus caused by D. phoenicicola in China. This disease severely delays the plant development of L. polystachyus and significantly reduces the yield and quality of sweat tea. Our findings will contribute to the control of brown spot of L. polystachyus.

First report of Stenocarpella macrospora causing Diplodia leaf streak disease of maize in Yunnan Province, China.

Maize (Zea mays. L) is cultivated globally as a staple food crop, animal feed, and biofuel. However, persistent diseases in maize have led significant yield losses and a decline in grain quality (Yang et al., 2017). In August 2024, long and narrow lesions were observed on the leaves of maize growing in a field in Luoping County, Qujing City, Yunnan Province, China (24° 94' 87" N, 101° 32' 24" E). The incidence of the disease was investigated to be about 15-30% in different varieties of maize planting areas in seven townships. Initial symptoms appeared as yellow to tan stripes, with the lesions measuring 3 to 48 mm in width and 45 to 560 mm in length. In severe cases, the lesions expanded, leading to complete leaf necrosis, which caused significant damage to maize production. Dark brown sub-epidermal pycnidia and conidia were observed on the necrotic leaf tissue. The pycnidia were globose or sac-like structures, with short and narrow conidiophores. The newly formed conidia are narrow, non-septate, and have transparent cytoplasm. The conidia are subcylindrical to narrowly ellipsoid, straight, curved, or occasionally irregular, measuring 38 to 86 × 7.2 to 12.3 µm. They are 0-3-septate, smooth-walled, pale brown, apex obtuse, base truncate. Therefore, 80 individual conidia were picked under a dissecting microscope, transferred to PDA plates containing antibiotics (10 mg/L rifampicin and ampicillin) followed by incubation at 28°C for 7 days (Fei et al., 2019). After seven days, the colonies of all isolates were turned grey and downy. Based on the maize leaf symptoms and the morphological characteristics of the pycnidia and conidia, the fungus was tentatively identified as Stenocarpella spp. (Luna et al., 2016). To confirm the identity of all isolates, DNA was extracted from 10 randomly selected isolates using the cetyl trimethyl ammonium bromide (CTAB) method. The internal transcribed spacer (ITS) region and the translation elongation factor 1α (TEF-1α) gene were amplified using the primer pairs ITS-1/ITS-4 and EF-1/EF-2, respectively, and then sequenced (Lamprecht et al., 2011). After comparison, the two target sequences of 10 isolates were consistent. BLASTn analysis revealed 99.27% and 100.00% homology with the two target sequences of Stenocarpella macrospora (syn. = Diplodia macrospora) (GQ259128.1, MG934504.1). Based on phylogenetic analysis of two targets, the isolate MS01 was clustered on the same evolutionary branch with S. macrospora. The two target sequences of isolate MS01 was submitted to GenBank under the accession numbers PQ241617.1 and PQ243292.1. To confirm the pathogenicity, a suspension (1 × 106 spores /mL) of isolate MS01 conidial was sprayed onto nine healthy leaves of maize plants in vitro and three pots cultivated with the plants (five plants per pot) in vivo. Plant treated with sterilized water served as control group. The treated plants were incubated at 28°C, 90% relative humidity with alternating light-dark cycles (i.e. 12 h shifts) for 3-10 days. At the same time, the conidial suspension was injected and inoculated onto maize ears, with water used as a blank control. Each treatment contained 3ears, 10 µL of the suspension was applied to each seed treatment. Inoculated leaves developed brown spots that gradually expanded into streak lesions extending toward the ends of the leaves, similar to those observed in the field. Additionally, the maize seeds become also moldy, whereas the control leaves and ears remained symptomless. The same pathogen was re-isolated from the infected leaves and ears, confirming Koch's postulates. Previously, S. macrospora and S. maydis have been reported in many countries across North, Central and South America, Africa, Asia, and Oceania, where maize is grown commercially. These pathogens are known to infect maize ears, stalks, and leaves of maize and also can produce mycotoxins (Romero et al., 2015; Snyman et al., 2011). To our knowledge, S. macrospora causing dry rot and ear rot of maize has been reported in China, but it is mostly found in quarantine at the time of importation (Tai et al., 1979; Shan et al., 2024). This is the first report of S. macrospora causing Diplodia leaf streak disease of maize in Yunnan Province, China. Since Stenocarpella spp. is one of the most destructive diseases of maize globally, the results of this study provide a foundation for developing effective prevention, control, and quarantine strategies for managing this disease.

Identification and Characterization of Bipolaris secalis and B. setariae Associated with Brown Stripe Disease on Saccharum officinarum in Yunnan, China.

Sugarcane (Saccharum officinarum L.) is the most important sugar crop that belongs to the Poaceae family and is mainly cultivated in tropical and subtropical regions worldwide. Brown stripe disease is a common and serious foliar fungal disease of sugarcane and has become a serious threat to sugarcane production in China. Yunnan Province is the second-largest sugar base in China. From 2022 to 2023, brown stripe-like symptoms from different sugarcane variety were observed in Yunnan. Thirty-nine Bipolaris-like isolates were obtained from symptomatic leaves of different sugarcane varieties and characterized by means of morphological identification combined with multilocus phylogeny comprising internal transcribed spacer rDNA (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the translation elongation factor-1α (TEF-1α) gene sequence date. The analysis provided strong support for the delineation and identification of two Bipolaris species associated with sugarcane brown stripe, including Bipolaris secalis and B. setariae. Pathogenicity text in potted sugarcane plants confirmed that these two Bipolaris species can cause brown stripe of sugarcane, thus fulfilling Koch's postulates. This study confirmed that B. secalis and B. setariae were the pathogens of brown stripe in Yunnan Province. To the best of our knowledge, this is the first to report B. secalis as causal agents of brown stripe of sugarcane. Collectively, this finding provides a basis for sugarcane brown stripe disease accurate diagnosis and would be helpful to the development of effective management strategies.

Isolation and identification of Cladosporium tenuissimum causing leaf blight on garden pea (Pisum sativum L.) in Telangana from southern India

Garden pea (Pisum sativum L.), is an important legume grown worldwide as a source of proteins and carbohydrates. Since pea crop is cultivated globally in cool temperate climatic conditions, increasing temperatures accompanied by high humidity favors fungal infections. A fungal infection causing leaf blight symptoms was frequently observed on the leaves of pea plants grown in greenhouse and field conditions. The blight symptoms showed a water-soaked and wilting appearance with curly leaf edges accompanied by greyish mycelial growth and chlorotic lesions. The disease incidence in field conditions ranged between 5 and 10 %, whereas in the controlled greenhouse conditions, it ranged from 40 to 45%. Two isolates of the fungus, Gp03 and Gp04 were isolated from Arkel pea leaves and purified from a single conidium each. According to Koch's postulates, the pathogenicity tests with these isolates using detached leaf assay and whole plant leaf assays confirmed the pathogen. Morphological and molecular analysis of nucleotide sequences of the D1-D2 region, translation elongation factor-1α (TEF) and actin (ACT) genes confirmed these isolates as members of the genus, Cladosporium. The phylogenetic relatedness of these isolates with other members of the Cladosporium genus revealed them as fungal strains of Cladosporium tenuissimum. Further, these isolates were also confirmed as C. tenuissimum by Microbial Type Culture Collection and Gene Bank (MTCC), Chandigarh in India, and provided the accession nos. MTCC 13581 and MTCC 13582 to the C. tenuissimum strains, Gp03 and Gp04 respectively. This is the first report of Cladosporium tenuissimum infecting garden pea in Telangana, India.

Phytosanitary problems in elephant garlic (Allium ampeloprasum var. holmense) in the “Val di Chiana” area (Central Italy), and evaluation of potential control strategies

Allium ampeloprasum var. holmense (elephant garlic) is traditionally cultivated in “Val di Chiana”, an area between Umbria and Tuscany regions of Central Italy, under the name “Aglione della Valdichiana”. This product has recently increased in importance, becoming a key economic resource for local farmers. In 2019, phytosanitary problems of elephant garlic cloves ready for transplanting emerged in this cultivation area. Symptom/sign observations and fungal isolations were performed for cloves divided into four components (tunic, basal plate, reserve tissue and shoot) from six farms in the “Val di Chiana” area. Isolates obtained were identified, using partial β-tubulin (BenA) and calmodulin (CaM) or translation elongation factor 1α (tef1α) genes sequences, as belonging to Penicillium [P. allii (95%), P. citrinum (4%), P. brevicompactum (1%)] or Fusarium [F. oxysporum (81%), F. proliferatum (19%)]. Fusarium spp. were mainly associated with clove tunics and basal plates, while Penicillium spp. with basal plates, reserve tissues and shoots. Fungi often also developed from asymptomatic components, but a correlation was found between isolated pathogens and disease symptoms. Pathogenicity and virulence towards elephant garlic cloves were verified for a representative isolate of each identified species, and Penicillium allii was the most virulent. Strategies to control Fusarium and Penicillium spp. on cloves were assessed, including chemicals, a biocontrol agent, surface sterilization and heat treatment. Among these, treatments with Patriot Gold® (active ingredient [a.i.] Trichoderma asperellum TV1, approved in organic farming on crops similar to elephant garlic), or Signum® (a.i. boscalid + pyraclostrobin, approved for Integrated Pest Management systems on crops similar to elephant garlic), were effective in simultaneous reduction of Penicillium spp. and Fusarium spp. Transplanting of asymptomatic cloves combined with the use of the above treatments showed promising effects for pathogens control, and to assist elephant garlic crop establishment.

First report of Fusarium solani causing Fusarium wilt of Bletilla striata (hyacinth orchid) in Zhejiang province of China.

Bletilla striata, a member of the family Orchidaceae, is a perennial herbaceous plant used in Chinese medicine. It is a commonly cultivated economic crop in the Yangtze River Basin provinces of China, as its roots are used to treat bleeding and inflammation. In Zhejiang province, Bletilla striata has a planting area of 1400 hectares with a total production of approximately 2.6×106 kg. In October 2021, over 40% of B. striata plants showed severe wilt in a traditional Chinese medicine plantation (ca. 10 ha) in Xianju City, Zhejiang Province, China. In July, leaf curling, crinkling, and leaf-edge browning of the diseased plants were first noticed in the field. Then, necrotic streaks gradually spread to the roots. Stems displayed chlorosis and withering and when they were cut vertically, symptoms such as vascular bundle discoloration, appeared. After October, the individual plants slowly wilted and died, their aboveground parts became filamentous, and the epidermis detached from the corm's fibrous roots. Diseased plants were easily removed as the corm root had fractured. White mycelia were clearly seen in the stem. Three symptomatic leaves and three stems were cut, their surfaces disinfected, and plated on potato dextrose agar (PDA). Six strains were subsequently isolated from all samples. Fungal colonies with white to cream-colored mycelia from all tissues appeared after 3 d of incubation at 26 °C. Pure cultures obtained after monospore isolation were examined for their morphological characteristics. The colonies grew rapidly, were fluffy and appressed, and had cottony white to pale cream coloration. Microconidia were hyaline, oval to reniform, with zero or one-septate (4.0-12.0 × 1.0-5.5 µm), and usually formed on elongated monophialidic conidiogenous cells. Macroconidia were wide, fusiform, or slightly curved with one or three septa (23.0-36.0 × 4.5-7.0 μm). Chlamydospores were spherical and were abundant on carrot agar (CA) medium within 2 wk. Fresh mycelia and conidia that grew at 26 ℃ for 7 d were collected from PDA plates. Next, DNA was extracted using the Ezup Column Fungi Genomic DNA Purification kit (Sangon Biotech, Shanghai, China). We amplified a portion of RNA polymerase II second largest subunit gene (RPB2) using primers 5f2/7cr (O'Donnell et al. 2010), the internal transcribed spacer (ITS) region using primers ITS1F/ITS4 (White et al. 1990), and the partial translation elongation factor-1α gene using primers EF1/ EF2 (O'Donnell et al. 1998) from the genomic DNA and sent the PCR amplicons for sequencing at Tsingke Biotechnology Co., Ltd., Wuhan, China. A BLAST search of the obtained sequences (GenBank accessions OP743920, OP913183, and OP913180) showed 99-100% homology with the respective sequences of the Fusarium solani reference isolate NRRL46702 (O'Donnell et al. 2008). Based on the morphological and molecular characteristics and BLAST search, the fungus was identified as F. solani (Leslie and Summerell 2006). Pathogenicity of the purified F. solani isolate was assessed by inoculateing a F. solani spore suspension of 1×106 conidia/mL (20 mL per seedling) on corm wounds made with a toothpick. Four inoculated and three non-inoculated seedlings (sterilized water as a negative control) were grown in a greenhouse at 26 °C under natural sunlight and covered with plastic bags to maintain humidity for 72 h. After 15 d, leaf browning on leaf edges, new leaf bases, and corm epidermis was observed. Symptoms, similar to those detected in the original sample, developed on the inoculated leaves, whereas the controls remained asymptomatic. Fusarium solani was successfully re-isolated from all four inoculated seedlings, and their identity confirmed by generating partial Tef1 and RPB2 sequences, thereby fulfilling the Koch's postulate. To our knowledge, F. solani has not been previously reported as a pathogen of B. striata.

Development of an RPA-based CRISPR/Cas12a assay in combination with a lateral flow strip for rapid detection of toxigenic Fusarium verticillioides in maize

Fusarium verticillioides is an important phytopathogenic fungus that poses a threat to maize yield and quality in global maize-growing regions by causing Fusarium ear and stalk rot. The fungus is known to produce fumonisins, which are toxic secondary metabolites and have been associated with high incidences of esophageal cancer. The FUM1 gene is responsible for producing a crucial polyketide synthase required for fumonisin biosynthesis and is present in all pathogenic strains of F. verticillioides. This study aims to develop a rapid and accurate detection assay for F. verticillioides based on the FUM1 gene, by utilizing Chelex-100 resin for DNA extraction, recombinase polymerase amplification coupled with CRISPR/Cas12a cleavage and lateral flow detection (RPA-Cas12a-LFD) assay. The developed RPA-Cas12a-LFD assay exhibited remarkable specificity for F. verticillioides, and the lowest limit of detection was 2 ag DNA of F. verticillioides. The entire diagnostic process was completed in just 73 min, including sample DNA extraction, RPA reaction, Cas12a cleavage, and result readout. Furthermore, RPA-Cas12a-LFD assay was found to be equivalent to single-spore isolation and partial translation elongation factor 1α gene (TEF-1α) sequencing in identifying diseased samples in the field. In summary, this accurate and portable detection equipment has great potential for detecting and recognizing F. verticillioides, especially in areas where sophisticated lab equipment is not available.

First report of Neopestalotiopsis clavispora causing cashew leaf blight disease in India.

Cashew (Anacardium occidentale) is an important commercial crop and highly prone to many biotic and abiotic stress. During March 2021, severe leaf blight symptoms were observed in Priyanka variety with 25-30% incidence grown under greenhouse nursery at ICAR-Directorate of Cashew Research (ICAR-DCR), Puttur (12º74'08.92"N; 75º22'97.22"E), Karnataka. Initial symptoms include small, irregular necrotic spots and later, the spots enlarged and covered major portion of the leaf lamina. In severe infection, leaves exhibited coalescing of spots leading to blight appearance. The infected leaves were randomly collected (n=5) and surface sterilized with 1% sodium hypochlorite for 1 min followed by three washes in sterile distilled water (SDW). Samples were plated on PDA plates amended with Rifampicin (40 mg/L) and kept for incubation at 25±2 oC for 5 days (12/12 h dark light period). A white-greyish, aerial, cottony mycelium on upper side with light yellow colour on the reverse side was consistently isolated. The black viscous acervuli were observed after 10-12 days of incubation. The conidia were fusiform, five-celled, versicoloured with three olivaceous brown median cells, two terminal hyaline cells, measured 23.3±2.12 - 28.33±2.7 x 3.6±0.8 - 4.28±0.78 µm (n=30). The apical cells had two to three flexuous, unbranched appendages, and basal appendage was solitary, tubular and unbranched. Morphological and cultural characteristics confirmed the pathogen as Neopestalotiopsis sp. (Maharachchikumbura et al. 2012). Further, two representative isolates (CLB_SCN1 & CLB_SCN2) were subjected for molecular characterization selected for molecular identification based on ITS-rDNA, tef-1α and tub2 gene sequences and phylogenetic analysis. Genomic DNA was isolated from 15 days old cultures and internal transcribed spacer (ITS) of ribosomal DNA (rDNA) (White et al. 1990), translation elongation factor 1α (tef-1α) gene (O'Donnell et al. 1998) and beta tubulin (tub2) using ITS1/ITS4, TEF1/TEF2 and Bt2a/Bt2b (Carbone and Kohn 1999; Glass and Donaldson 1995) were amplified using primer pairs respectively. PCR amplicons were sequenced, and the sequences were deposited in GenBank (accession numbers: ITS: OP880881.1, OP880882.1; tef-1α: OP882579.1, OP882580.1; and tub2: OP882581., OP882582.1). The phylogeny was constructed based on combined ITS, tef-1a, and tub2 regions. Neighbour-Joining (NJ) analysis was conducted and the tree was constructed with the substitution models (branch support was evaluated by 1,000 bootstrap replications). Combined phylogeny confirmed that the sequences shared a common clade with N. clavispora. Hence, morphological, microscopic and molecular characterization confirmed the pathogen as N. clavispora. The pathogenicity test was done on six months old healthy grafts of Priyanka variety (n=9) and repeated thrice. Conidial suspension (2×106 spores/ml) of N. clavispora CLB_SCN1 (15 days old culture) was sprayed on the healthy cashew seedlings, and kept in greenhouse by covering with polythene bags for 24 h (>80 % RH) and maintained under greenhouse condition. The control grafts were inoculated with SDW. The inoculated plants showed blight symptoms after 7-10-day post inoculation and control remained heathy. Re-isolation was done from the symptomatic leaves and identity was confirmed using cultural and molecular studies. Earlier reports showed that, N. clavispora has been reported to cause cardamom leaf blight (Biju et al 2018) and leaf spot disease of plum (Banerjee and Rana 2020). To best of our knowledge, this is the first report of cashew leaf blight disease caused by N. clavispora from India (Farr and Rossman, 2022). Early detection will help farmer in better management and avoiding economic loss caused by N. clavispora.

First Report of Cytospora azerbaijanica Causing Cytospora Canker and Shoot Dieback on Peach (Prunus persica) in California, U.S.A.

Peaches (Prunus persica L.) are an important crop in the United States with California leading the nation in peach production, with approximately 505,000 tons valued at $378.3 million (USDA National Agricultural Statistics Service, 2021, https://www.nass.usda.gov/). From April to July 2022, symptoms of branch and scaffold canker as well as shoot dieback were observed in three peach (cvs. Loadel, Late Ross and Starn) orchards located in San Joaquin County, California. Samples were collected from about 12 trees for each cultivar. Fast-growing, white, flat colonies were consistently isolated from active cankers on acidified potato dextrose agar (APDA) following the method described by (Lawrence et al. 2017). Pure fungal cultures were obtained by transferring single hyphal tips onto new APDA Petri plates. A total of 22 isolates were obtained. Each fungal isolate was recovered from a single diseased branch (40 to 55% recovery). All isolates in this study shared similar morphological characteristics. Fungal colonies were fast-growing with relatively even but slightly dentate margin, flat with white to off-white mycelium that turned vinaceous buff to pale greyish sepia (Rayner 1970) with age. Black, globose, ostiolated pycnidia, 0.8-(1.3)-2.2 mm diameter, with brownish surface hyphae formed on peach wood embedded in PDA after approximately three weeks and exudated buff-colored mucilage. Pycnidia were both solitary and aggregated and had multiple internal locules sharing invaginated walls. Conidiogenous cells were hyaline, smooth-walled, septate, tapering towards the apex, 13-(18.2)-25.1 × 0.8-(1.3)-1.9 µm (n = 40). Conidia were hyaline, allantoid, smooth, aseptate, 5.5-(6.3)-7.1 × 1.4-(1.9)-2.3 µm (n = 40). Genomic DNA was extracted and sequences of the internal transcribed spacer region (ITS) using ITS5/ITS4 universal primers, translation elongation factor 1α gene (TEF) using primers EF1-728F/EF1-986R, second largest subunit of RNA polymerase II (RPB2) using primers RPB2-5F2/fRPB2-7cR, and actin gene region (ACT) using primers ACT-512F/ACT-783R were obtained and compared with sequences available in GenBank (Lawrence et al. 2018; Hanifeh et al. 2022). Isolates were identified as Cytospora azerbaijanica following DNA sequencing and morphological identification. Consensus sequences of the four genes of two representative isolates (SJC-66 and SJC-69) were deposited into GenBank database (ITS: OQ060581 and OQ060582; ACT: OQ082292, OQ082295; TEF: OQ082290 and OQ082293; RPB2: OQ082291 and OQ082294). The Basic Local Alignment Search Tool (BLAST) indicated that the sequenced RPB2 genes of isolates (SJC-66 and SJC-69) were at least 99% identical to that of Cytospora sp. strain shd47 (Accession: MW824360) covering at least 85% of the sequences. The actin genes from our isolates were at least 97.85% identical to that of Cytospora sp. strain shd47 (Accession: MZ014513), covering 100% of the sequences. The translation elongation factor gene from isolates (SJC-66 and SJC-69) was at least 96.4% identical to that of Cytospora sp. strain shd166 (Accession: OM372512), covering 100% of the query. Those top hit strains belong to C. azerbaijanica, recently reported by Hanifeh et al. (2022). Pathogenicity tests were performed by inoculating eight wounded, 2- to 3-year-old healthy branches on each of eight 7-year-old peach trees, cvs. Loadel, Late Ross and Starn, using 5-mm-diameter mycelium plugs collected from the margin of an actively growing fungal colony on APDA. Controls were mock-inoculated with sterile agar plugs. Inoculation sites were covered with petroleum jelly and wrapped with Parafilm to keep moisture. The experiment was performed twice. After four months, inoculation tests resulted in vascular discoloration (canker) above and below the inoculation sites (average necrosis length of 114.1 mm). Cytospora azerbaijanica was re-isolated from all infected branches (70 to 100% recovery) completing Koch's postulates. Controls remained symptomless and no fungi were isolated from the slightly discolored tissue. Cytospora species are destructive canker and dieback pathogens of numerous woody hosts worldwide. Recently, C. azerbaijanica was reported in causing canker disease of apple trees in Iran (Hanifeh et al. 2022). To our knowledge, this is the first report of C. azerbaijanica causing canker and shoot dieback of peach trees in the United States and worldwide. These findings will aid towards a better understanding of genetic diversity and host range of C. azerbaijanica.

First report ofFusarium meridionalecausing Fusarium head blight of wheat in Henan Province, China.

Fusarium graminearum and F. asiaticum have been found as a major cause of Fusarium head blight (FHB) of wheat (Triticum aestivum L.), especially in Henan Province of China (Zhang et al. 2014; Xu et al. 2021). In May 2021, a survey to determine the composition of Fusarium species infecting wheat heads was conducted in commercial fields in Henan. A total of 395 diseased spikes with premature whitening symptom were collected from 31 commercial fields in Henan. Symptomatic spikelets were excised, surface-sterilized for 10 s in 70% ethanol followed by 1 min in 3% sodium hypochlorite, rinsed three times with autoclaved distilled water, and then plated onto potato dextrose agar (PDA) medium. Isolated colonies that resembled Fusarium species were transferred to fresh PDA plates and purified using a single spore method. Species were identified based on sequence analysis of the translation elongation factor-1α (TEF) and trichothecene 3-Oacetyltransferase (Tri 101) gene (Proctor et al. 2009). The results indicated that F. graminearum (43.3%), F. asiaticum (47.8%), F. pseudograminearum (6.6%) were the main causal agents of FHB in Henan. However, nine isolates (2.3%) were found to be identical to F. meridionale by sequence comparison in GenBank, and eight isolates of which came from three fields with 1% to 2% diseased spikes near Reservoir Luhun (34.1255° N, 112.1111° E, altitude: 388 m above sea level), Songxian County of Henan. The isolates of F. meridionale were transferred onto carnation leaf agar (CLA) and incubated at 20℃ under black light blue illumination. Macroconidia were abundant, relatively slender, curved to almost straight, commonly six- to seven-septate, and 27.0 to 61.0 (average 44.0) μm × 3.2 to 6.8 (average 5.3) μm. Microconidia were not observed. The TEF sequences (Accession nos. OM460748 to OM460756) and the Tri 101 sequences (OM460759 to OM460767) of the nine isolates showed 99 to 100% similarity with the TEF and Tri 101 sequences of F. meridionale NRRL 28436 and NRRL 28723 (AF212435 and AF212436 (TEF); AF212582 and AF212683 (Tri 101)). To complete Koch's postulates, the pathogenicity of the fungus was tested by using the single floret inoculation method by injecting 20-μl conidial suspension (5 × 105 conidia per milliliter) into healthy inflorescences of wheat cultivar Bainong 207 at anthesis in the field. Another 30 healthy inflorescences were injected with sterile distilled water. The heads were covered with polyethylene bags that were removed after 2 days. Twenty days after inoculation, while control inflorescences were asymptomatic, the F.meridionale-inoculated inflorescences showed 12% bleached spikelets per spike. By using the methodology described above, the fungus was re-isolated from infected spikelets of inoculated wheat heads but not from the controls. Although F.meridionalehas frequently been reported in association with Fusariumear rot (FER) of maize in Chongqing City and Gansu Province (Zhang et al. 2014; Zhou et al. 2018), and riceFER in Sichuan Province (Dong et al. 2020), to our knowledge, this is the first report of F. meridionale from diseased wheat heads in Henan, China. Further investigation is needed to gain a better understanding of this species by collecting isolates from different cropping system in Henan, which maize-wheat and rice-wheat rotation fields have coexisted in the region.

First report of Fusarium solani causing wilt disease on Gleditsia sinensis in Tianjin, China.

The Gleditsia sinensis Lam. widely grown in China is a perennial plant with medicinal properties (Zhang et al. 2016). Since 2019, the leaves of G. sinensis have exhibited yellowing and wilting, and the plants have gradually become stunted and dead in Taifeng park of Binhai New Area in Tianjin (39.02° N; 117.65° E). In this park, there are two types of G. sinensis, one is with round branch thorns, the other is with flat branch thorns. The G. sinensis with round branch thorns did not grow well and almost all plants had disease symptoms. The samples were collected on October, 2021 and deposited in Plant Disease Laboratory of Tianjin Agricultural University under accession no. PATAU211018. The disease symptoms consisted of foliage wilt (Figure 1A), plant drying and vascular tissue discoloration (Figure 1B). The stem sections from different plants were surface-disinfested in 0.6% NaClO, wiped with 75% ethanol and rinsed with sterile water. Thirty tissue samples were placed on Potato Dextrose Agar (PDA) medium and cultured at 28℃ for 7 days (Uppala et al. 2013). Thirty fungal isolates with the same morphological characteristics were obtained from the samples. Five representative isolates (PATAU211018-05, PATAU211018-07, PATAU211018-10, PATAU211018-12 and PATAU211018-21) were collected and purified using the single-spore method (Li et al. 2022). Colonies of the five isolates on PDA grew in a circular shape and showed abundant white densely fluffy aerial mycelium (Figure 1C). Morphological characteristics included septate and hyaline hyphae, long cylindrical monophialides (Figure 1D), macroconidia (Figure 1E) and microconidia (Figure 1F). Macroconidia were falcate, 2-5-septate, hyaline, 18-40 × 4-6 μm (n = 50). Microconidia were hyaline, oblong, 0-1-septate, 5-14 × 2-6 μm (n = 50). These morphological characteristics were consistent with the description of Fusarium solani. (Chitrampalam et al. 2018). PATAU211018-12 was randomly chosen for molecular analysis as the representative isolate given the similarity of these isolates. For further identification, the genomic DNA of isolate PATAU211018-12 was extracted. The fragments of internal transcribed spacer (ITS), translation elongation factor 1α (EF1α) gene and RNA polymerase II subunit (RPB2) were amplified and sequenced (O'Donnell et al. 2008; Carbone et al. 1999). The sequences of ITS, EF1α, and RPB2 of PATAU211018-12 were deposited in GenBank under the accession no. of OP735578, ON630412 and OP746032, respectively. Phylogenetic trees were constructed in MAGA X (Kumar et al. 2018) using the neighbor-joining (NJ) method based on the concatenated sequences of ITS, EF1α, and RPB2 (Figure 2). The isolate (PATAU211018-12) grouped with F. solani (JS-169) with a bootstrap value of 100 in the phylogenetic tree. The morphology and multi-gene phylogenetic analysis indicated that the new isolate is F. solani. Pathogenicity tests were carried out on one-year-old G. sinensis seedlings with round branch thorns (n=6). The F. solani isolate PATAU211018-12 was cultured in Potato Dextrose Broth (PDB) at 28°C on a shaker at 150 rpm for 5 days. Mycelia were filtered through four layers of sterilized lens paper and the conidia were obtained for pathogenicity tests. G. sinensis was infected by F. solani through root soaking method. The roots were inoculated by dipping in conidial suspension with the concentration of 107 conidia/mL for 30 minutes. Control plants (n=6) were treated with distilled water. Plants were in pots indoors at 25℃. At 20 days after inoculation, the leaves of inoculated plants were chlorotic and wilted (Figure 1G), symptoms similar to those observed in the park. In contrast, the leaves of control plants were symptomless (Figure 1H). The pathogenicity assay was repeated three times. The fungal isolate was re-isolated from the disease tissues and verified as F. solani based on morphology and molecular character (ITS, EF1α and RPB2). F. solani has been reported as pathogens on many plants, such as Eriobotrya japonica (Wu et al. 2021), Fragaria × ananassa (Pastrana et al. 2014), Gastrodia elata (Li et al. 2022) and Hedysarum boreale (Uppala et al. 2013). To our knowledge, this is the first report of F. solani causing disease on G. sinensis in China. Identification of F. solani as a disease agent in G. sinensis will assist in disease management for this important tree crop.

Fusarium diversity from the Golden Gate Highlands National Park.

Members from the genus Fusarium can infect a broad range of plants and threaten agricultural and horticultural production. Studies on the diversity of Fusarium occurring in natural ecosystems have received less attention than the better known phytopathogenic members of the genus. This study identified Fusarium species from soils with low anthropogenic disturbance found in the Golden Gate Highlands National Park (GGHNP), a part of the Drakensberg system in South Africa. Selective techniques were implemented to obtain 257 individual isolates from the selected soil samples for which the translation elongation factor 1α (tef-1α) gene region was sequenced and compared against the Fusarium MLST and FUSARIUM-ID databases. Phylogenetic analyses, based on maximum likelihood and Bayesian inference, were used to determine species diversity in relation to reference isolates. Species level identifications were made within three of the seven species complexes and identified F. brachygibbosum, F. sporotrichioides, F. andiyazi, and F. gaditjirri based on the FUSARIUM-ID database, with F. transvaalense and F. lyarnte identified against the Fusarium MLST database. This indicated highly diverse populations of Fusarium from soils with low anthropogenic disturbance from the Afromontane grassland region found in mountain ranges.

First Report of Phytophthora occultans Causing Dieback of Buxus sempervirens in the Czech Republic.

In Nov 2011, and then recurrently since Sep 2020, an extensive decline has been recorded in boxwood (Buxus sempervirens), sometimes with several dozens of damaged individuals planted in private gardens and public areas and purchased in amateur markets in the Czech Republic. The leaves of the plants first showed orange-bronze discoloration, then dried and fell off, and the affected plants died. The roots, collars and stems of these plants had dark brown to black necrotic lesions. Phytophthora occultans Man in 't Veld & K. Rosend. was consistently isolated on selective medium PARPNH (Jung et al. 1996) directly from segments of symptomatic collar tissues and from rhododendron leaf pieces used to bait excised roots. On 20% V8 agar (V8A) and on carrot agar (CA), colonies had a stellate pattern. Radial growth at 25°C was 9.4 mm/day on V8A and 5.3 mm/day on CA. The cardinal growth temperatures were min. 7°C, optimum 25 to 27°C, and max. 32°C. The isolates were homothallic and produced on CA colorless globose oogonia ranging from 25.4 to 36.4 µm (n = 40) in diam. Oospores were slightly aplerotic and measured (n = 40) 22.5 to 31.9 µm in diam., with a 0.9 to 1.5 µm thick wall. The antheridia were predominantly paragynous and averaged 11.5 × 9.9 µm (height × width, n = 40). Noncaducous sporangia were obpyriform, ovoid, elongated to irregular and semipapillate, sometimes bipapillate and measured (n = 40) 31.4 to 73.4 × 17.8 to 32.1 µm, and the L:B ratio was 1.9 to 2.0. Chlamydospores and hyphal swellings were not observed. The morphological characteristics resembled those described for P. occultans (Man in't Veld et al. 2015). The isolates were deposited in the Czech Collection of Phytopathogenic Oomycetes (CCPO) under accession nos. 551.11, 1158.20, 1176.21, 1201.21, 1218.21, 1236.21 and 1261.22. For molecular identification, the internal transcribed spacer (ITS) region, cytochrome oxidase subunit 1 gene (COX1), and translation elongation factor-1α (EF) gene from all isolates were amplified and sequenced using the primer pairs ITS4/ITS5 (White et al. 1990), COXF-CIT/COXR-CIT (Man in't Veld et al. 2015), and ELONGF1/ELONGR1 (Kroon et al. 2004), respectively. The resulting sequences of representative isolates P1158.20 and P1176.21 were deposited in GenBank (accession nos. MW750576 and OP326036 for ITS, ON862131 and OP313505 for COX1 and MW762616 and ON862132 for EF). BLASTn searches of GenBank, using the partial ITS, COX1, and EF sequences, revealed 100, 100, and 99% sequence identity, respectively, to P. occultans ex-holotype culture CBS101557 accessions JX978155, JX978156 and KF650770 (Man in't Veld et al. 2015). Concatenated sequences of the three genes were used to conduct a phylogenetic analysis using the maximum likelihood method in MEGA 11 (Tamura et al. 2021). The isolates were identified as P. occultans based on morphology and a multigene phylogenetic analysis. Koch´s postulates were confirmed by a soil infestation test. Healthy 2-year-old B. sempervirens plants were inoculated (9 plants per isolate and control, isolates no. 1158.20, 1176.21, 1261.22) with three 5-mm-diam. V8A mycelial plugs by inserting into the substrate near the collar. Control plants were treated with sterile agar plugs. All plants were kept in a greenhouse at 25°C and exposed to 24 h of flooding up to collar once a week. All inoculated plants showed wilting, collar lesions and root rot occurred after 21 days, while control plants remained healthy. The pathogen was reisolated from infected plants and confirmed by molecular identification. P. occultans was found for the first time in 1998 on Buxus sempervirens in the Netherlands and later in Belgium, the United Kingdom, Germany and Romania (Man in´t Veld et al. 2015, Nechwatal et al. 2014), as well as in the USA (Reeser et al. 2015, Gitto et al. 2018). This is the first report of P. occultans in the Czech Republic. This pathogen likely poses another significant threat to boxwood cultivation in addition to the previously invaded Cydalima perspectalis and Calonectria pseudonaviculata.

First report of Calonectria ilicicola causing fruit rot on postharvest Prunus persica in Zhejiang Province, China.

Peach (Prunus persica) is an important economic tree fruit in China, with 15 million tons produced in 2020 (Xu et al. 2022). In September 2021, fruit rot on postharvest P. persica 'Yingqingtao' was observed in an orchard warehouse in Qixing district (120°41'E, 29°15'N), Zhejiang Province. Disease incidence was estimated at 25%, and yield loss was estimated at approximately 20% of the total yield. The naturally infected fruit had water-soaked, light brown lesions that fused, and produced a gray-white, dense mycelium (Fig. 1 A). The mycelia were transferred using a sterilized toothpick to potato dextrose agar (PDA) and cultured for 7 d. Macroconidia were used to produce five single-spore isolates, each from a different fruit. Six-day-old colonies grown on PDA at 26°C had light brown centers with gray-white edges; on the underside the centers were reddish brown and white towards the margin (Fig. 1 D). Isolate TGF2 was selected for further identification. Macroconidia were hyaline, straight, cylindrical, and one-to-three septae, 63.2 to 81.8 × 5.7 to 7.8 μm (mean = 73.9 ± 4.3 × 6.9 ± 0.5, n = 30) (Fig. 1 E). Chlamydospores were produced abundantly on PDA (Fig. 1 F), and measured 11.7 to 19.4 × 8.5 to 16.9 μm (n = 10). Perithecia were reddish orange, globose, and 329.9 to 417.1 μm in diameter on PDA (Fig. 1 G). Asci were hyaline and clavate, 61.2 to 91.8 × 14.4 to 20.7 μm (n = 10); ascospores were hyaline, slightly curved, 1- to 3-septate, mostly 1-septate, and 37.6 to 59.7 × 4.9 to 6.4 μm (mean = 49.9 ± 4.5 × 5.6 ± 0.4, n = 30) (Fig. 1 H-J). Morphological characteristics placed this organism within the Ca. kyotensis species complex (Liu et al. 2020). For molecular identification, the internal transcribed spacer (ITS: OP164807-OP164811), calmodulin (Cal: OP176049-OP176053), histone3 (His3: OP176054-OP176058), and translation elongation factor 1α (Tef1: OP176044-OP176048) genes were sequenced (Liu, et al., 2020). The twenty sequences were deposited in GenBank. A BLAST search of these sequences showed 99% identity with sequences of the ex-holotype Ca. ilicicola CMW 30998 (Liu et al. 2020). Bayes phylogenesis suggested that these strains and Ca. ilicicola CMW 30998 were clustered in the same clade (Bayesian posterior probability = 1) (Fig. 2). Integrating morphology and molecular data, these strains were identified as Ca. ilicicola. For pathogenicity tests, P. persica fruits were surface sterilized in 75% ethanol for 30 s and air-dried for 5 mins to allow the alcohol to volatilize. A conidial suspension (30 mL of 1 × 106 conidia/mL) of TGF2 was sprayed onto ten fruits, and ten fruits sprayed with sterilized water served as controls. The experiment was repeated three times. Fruits were kept on a mist bench at 26°C and 60% relative humidity. After 5 days, inoculated fruits showed necrotic lesions and a dense, gray-white mycelium, however, the control fruits showed no symptoms (Fig. 1 B, C). Ca. ilicicola was reisolated from lesions of inoculated fruits. Ca. ilicicola has been reported from Vaccinium sp., Glycine max, Medicago sativa (Farr and Rossman 2022; Kleczewski et al. 2019; Zhang et al. 2020). To our knowledge, this is the first report of Ca. ilicicola causing fruit rot of P. persica in China. In other research on Ca. ilicicola, we found that continuous light could inhibit its growth, suggesting a method to protect postharvest peaches.

Identification and First Report of Leaf Blight Caused by Phytophthora palmivora on Chinese Water Bamboo in China.

Chinese water bamboo (Dracaena sanderiana) is a popular houseplant due to its ability to survive in various indoor conditions. In October 2020, pronounced leaf blight symptoms with approximately 50% disease incidence were observed on water bamboo in the 35-ha field of Wenchang county (19°50'45'' N, 110°21'38'' E), Hainan, China. The diseased leaves showed pale green with yellowish lesions, dried and shrivelled tips. As the disease progressed, upward-spreading necrosis led to stem weakness, and then the plants wilted and died within a few weeks (Fig. 1A,B). Ten symptomatic leaves were collected, and leaf pieces (5 mm × 5 mm) from the edge of the lesion were cut, disinfected for 30 s using 75% ethanol, and rinsed five times in d.d. H2O, and placed on V8 media amended with 10 mg/L pimaricin, 150 mg/L ampicillin, and 16 mg/L rifampicin (Guo et al., 2012). Eight isolates of Phytophthora recovered from 10 leaves were characterized morphologically. Colony morphology showed slightly radiate to stellate patterns, with cottony aerial mycelium (Fig.1D). Hyphae were smooth and uniform, branching at nearly right angles into stout (Fig.1E). Mycelial swellings were observed in plate culture (Fig.1F). Chlamydospores were abundant and spherical (Fig.1G). Sporangia were produced externally through sympodial development of the sporangiophore immediately below a sporangium (Fig.1H, I). The sporangia were 29.3-63.9 µm (mean, 48.5 µm) in length and 19.3-46.8 µm (mean, 33.5 µm, n=50) in width. No sexual organs were observed in the 8 isolates. The morphological characteristics of the isolates were consistent with the description of P. palmivora (Erwin and Ribeiro, 1996). The internal transcribed spacer (ITS) region of rDNA, the translation elongation factor 1α gene (TEF1), and β-Tubulin gene (TUB) sequences of the 8 isolates were amplified using the primers ITS1/ITS4 (Cooke et al., 2000), ELONGF1/ELONGR1 and TUBUF2/TUBUR1 (Kroon, et al., 2004), respectively, followed by sequencing analysis. The sequences of representative isolate HK-5 for the ITS, TEF1 and TUB were deposited in GenBank with the accession numbers of OK349485, OK349488 and OK349487. BLAST results showed that the ITS (MG865559, identity=735/735; 100%), TEF1(MH359047, identity=682/684; 99.7%) and TUB (MH493992, identity=467/468; 99.8%) sequences were all highly similar with a sequence of P. palmivora strain CPHST BL105. Phylogenetic analysis using the combined ITS, TEF1, and TUB sequences showed that the isolate HK-5 were grouped with the P. palmivora with good bootstrap support (Fig.2). Based on morphological and molecular identification, the pathogens were identified as P. palmivora. The pathogenicity tests showed that all whole plants (1-year-old) sprayed with the 5 mL of zoospore suspension (1×106 zoospores mL-1) initially presented with discoloured spots on their leaves after 4 days of inoculation, and the symptoms gradually progressed from spots to leaf blight (Fig. 1C). Each isolate was applied onto 10 plants and control plants were sprayed with d.d.H2O. The results of the pathogenicity test exhibited typical symptoms as observed in the field. No significant differences of virulence were observed among the 8 isolates, and the control plants remained symptomless. P. palmivora was re-isolated from the leaves of inoculated plants. This is the first report of P. palmivora on water bamboo in China, and appropriate measures must be undertaken to control this agent in this region.

First Report of Diaporthe phoenicicola causing leaf spot on blueberry (Vaccinium virgatum) in China.

Blueberry (Vaccinium virgatum), a member of the Ericaceae family, is an increasingly important crop in China because of its abundant nutritional benefits and economic value (Kuzmanović et al. 2019). In October 2021, leaf spots were detected on 'Rabbiteye' blueberry at the Agricultural Science and Technology Park of Jiangxi Agricultural University in Nanchang, China (28°45'51"N, 115°50'52"E), which caused severe defoliation of the crop and fruit yield losses of 25% (Figure 1A). Disease surveys were conducted at that time; the results showed that disease incidence was 75.5%, observed in 151 of the 200 accessions sampled, and this disease had not been found at other cultivation fields in Nanchang. Lesions with taupe to dark brown margins were irregularly shaped and associated with leaf margins. Spots coalesced to form larger lesions, with black pycnidia present in more mature lesions. To identify the causal agent, 10 small pieces (5 mm2) of leaf tissue excised from the lesion margins were surface sterilized in 75% ethanol solution for 30 s and 0.1% mercuric chloride solution for 2 min, rinsed three times with sterile distilled water, then placed on potato dextrose agar (PDA) at 25°C for 5 to 7 days in darkness. Five fungal isolates showing similar morphological characteristics were obtained as pure cultures by single-spore isolation. All fungal colonies on PDA were floccose, dense, and white (Figure 1B.C). Black pycnidia developed on PDA at 25°C under a 12/12 h light/dark cycle for 30 days. Alpha conidia were 6.17 to 8.53 × 1.64 to 3.20 µm (average 6.94 × 2.52 µm, n = 100), aseptate, hyaline, fusiform to ellipsoidal, often biguttulate. Beta conidia were 15.26 to 25.41 × 0.92 to 1.40 µm (average 20.14 × 1.27 µm, n = 30), aseptate, hyaline, linear to hamate (Figure 1D). Based on morphological characteristics, the fungal isolates were suspected to be Diaporthe spp. (Gomes et al. 2013). To further confirm the identity of this putative pathogen, two representative isolates (LGM1 and LGM2) were selected for molecular identification. The internal transcribed spacer region (ITS), translation elongation factor 1α (EF1-α), histone H3 (HIS), calmodulin (CAL), and β-tubulin (TUB2) genes were amplified from gDNA and sequenced using primers ITS1/ITS4 (Peever et al. 2004), EF1-728F/EF1-986R and CAL228F/CAL737R (Carbone et al. 1999), CYLH3F/H3-1b (Crous et al. 2004), Bt2a/Bt2b (Glass and Donaldson 1995), respectively. GenBank accession numbers of isolate LGM1 and LGM2 were OM778771 to 72 for the ITS region, OM868228 to 29 for EF1-α, OM837771 to 72 for TUB2, ON206971 to 72 for CAL, ON206973 to 74 for HIS. BLAST results showed that the ITS, EF1-α, TUB2, HIS, and CAL sequences showed 99% (538/545 bp), 100% (322/322 bp), 99% (480/484 bp), 99% (459/460 bp), 99% (430/433 bp) identity, respectively, with those of Diaporthe phoenicicola (GenBank accession no. MW504735, MW514099, MW514142, MW514067, MT409304). Two maximum likelihood phylogenetic trees were built based on the sequences of ITS, EF1-α, HIS, CAL, and TUB2 by using MEGA 5. The two isolates LGM1 and LGM2 clustered with D. phoenicicola (Figure 2 and 3). The fungus was identified as D. phoenicicola by combining morphological and molecular characteristics. To evaluate the pathogenicity, three healthy young potted V. virgatum plants were spray inoculated with a conidial suspension of 106 conidia/ml. Another set of three plants that were sprayed with sterilized distilled water served as the controls. The experiment was repeated three times, and all plants were maintained in a climate box (12 h light/dark) at 25°C with 80% relative humidity. Five days after inoculation, no symptoms were observed on control plants (Figure 1F), and all inoculated plants showed symptoms (brown flecks) similar to those observed in the field (Figure 1E). The fungus was reisolated from the infected tissues and confirmed as D. phoenicicola by morphological and molecular identification, and could not be isolated from the controls, fulfilling Koch's postulates. To our knowledge, this is the first report of D. phoenicicola causing leaf spot on blueberry in China. The discovery of this new disease and the identification of the pathogen will provide useful information for developing specific control measures and potential sources for resistance to this disease caused by D. phoenicicola.

Diagnosis and Control of Brown Leaf Spot of Kiwi (Actinidia deliciosa) Using Biochar-Zinc Oxide Nanocomposite (MB-ZnO) as a Non-Toxic Bio-Fungicides

Kiwi is one of the best natural sources of vitamin C and has wide applications. During October–November 2021, small brown spots were examined on the Kiwi leaves. The diseased leaf samples were collected and placed on potato dextrose agar nutrient media for diagnosis. Morphological, anatomical, and molecular studies revealed this disease-causing agent to be Rhizopus oryzae. Molecular characterizations of the isolated pathogen were performed by using actin translation elongation factor (EF-1α) and ribosomal deoxyribose nucleotide inter transcribed sequence (rDNA ITS ITS1/ITS4) and elongation factors (EFl-F/EFl-R) primers. A BLAST study of the resultant ITS1/ITS4 sequence showed > 99% resemblance with R. oryzae (MT603964.1), while the EF-1α sequence revealed 100% similarity with translation elongation factor-1α gene of R. oryzae (MK510718.1). The obtained ITS1/ITS4 sequence was submitted to NCBI (MW603842.1). Koch’s postulates established the pathogenicity of isolated R. oryzae and proved it to be the brown spot pathogen of Kiwi. For the environmentally-friendly management of Kiwi leaf spot, maize biochar-Zinc Oxide (MB-ZnO) nanocomposite was used. The prepared nanocomposite was characterized by Fourier transform infrared (FTIR) spectroscopy, thermo gravitational analysis (TGA), X-ray diffraction (XRD) analysis, scanning electron microscopy (SEM), and energy dispersive X-ray (EDX) analysis. After successful preparation, MB-ZnO was assessed for its possible antifungal potential against R. oryzae. MB-ZnO displayed substantial growth inhibition, and the highest growth inhibition (79%) was observed at a 19 mg/mL dose rate of nanoparticles. These excellent findings propose that Ball-milled synthesis is a fast, economical, and environmentally friendly method for nanocomposite in the near future. The nanocomposite is used as a nominal substitute for chemical fungicides.

First report of Gnomoniopsis smithogilvyi causing chestnut brown rot on nuts and burrs of sweet chestnut in Spain.

About 60% of the nut production of sweet chestnut (Castanea sativa Mill.) in Europe originates in Spain (FAOSTAT, 2022), mostly (91%) in Galicia (NW Spain). In September 2021, premature fall of immature chestnut burrs and nuts of C. sativa was observed in eight orchards of Pontevedra and Ourense (provinces of Galicia). Chestnut green burrs had turned brown and fallen off, and the nuts showed brown lesions in kernels and embryos. Some nuts had become mummified. Symptomatic samples of burrs and nuts, including mummified fruits, were collected. Small pieces of samples were surface-disinfected with 2% sodium hypochlorite, and plated onto potato dextrose agar (PDA) plates. Colonies were creamy white to gray or light brown, and presented a woolly to felty mycelium with a dense development in concentric circles. Brownish to black conidiomata, produced abundantly, were globose to sub-globose. Conidia were hyaline, oval, obovoid, fusoid and multi-guttulate, and 6.6±0.78 [5.07 to 9.01] μm x 3.2±0.43 [2.41 to 4.38] μm in size. These features matched those described for Gnomoniopsis smithogilvyi (Shuttleworth et al. 2012), syn. G. castaneae (Visentin et al. 2012; Shuttleworth et al. 2015). Genomic DNA was extracted from mycelium developed in 22 burrs and nuts, and 30 pure cultures. The rDNA internal transcribed spacer (ITS), fragments of the β-tubulin (TUB2) and the translation elongation factor-1α (TEF-1α) genes were amplified using ITS1F (Gardes and Bruns 1993) and ITS4 (White et al. 1990), T1/Bt2b (O'Donnell and Cigelnik 1997), and EF1-728F/EF1-986R (Carbone and Kohn 1999) primers, respectively. Two isolates (EFA 924A, EFA962.4A) were deposited in the Spanish Type Culture Collection (Paterna, Valencia), and their sequences submitted to GenBank (accession nos.: ITS: OM319846, OM319848; TUB2: OM417078, OM417080; TEF-1α: OM417081, OM417083). BLASTn analyses showed: for ITS and TEF-1α sequences, ≥99.7% identity to the ex-type of G. smithogilvyi (ITS: JQ910642, 474 matching bp; TEF-1α: JQ910645, 335-338 matching bp), and for TUB2 sequences, ≥99.1% identity to G. castaneae (LN999975, 446-453 matching bp). Pathogenicity tests were carried out on surface disinfected nuts of Castanea sativa `Raigona´. A superficial wound was made in the pericarp of each nut. A 2-mm mycelial plug of a 7-days-old culture of G. smithogilvyiwas then inoculated: twenty nuts with isolate EFA924A and twenty with isolate EFA962.4A. Twenty nuts inoculated with sterile agar were used as control. The holes were sealed with Parafilm®. Nuts were incubated in a moist chamber at 22±2°C. Two replicated tests were carried out. Four inoculated and four control nuts were inspected for the presence and progress of rot symptoms every seven days. Three weeks after inoculation, all remaining inoculated nuts were completely rotted, whereas all control nuts remained healthy. Gnomoniopsis smitholgilvyi was reisolated from all inoculated nuts, and it was not recovered from the controls. This pathogen causes chestnut brown rot on sweet chestnut worldwide (EPPO, 2022), causing also shoot cankers on chestnut (Lione et al, 2019). This is the first report of G. smithogilvyi causing chestnut brown rot on nuts and burrs of C. sativa in Spain. Future studies on the incidence of this pathogen and its impact on chestnut yield should be carried out in the main European producing countries, i.e. Spain, Italy, Portugal and Greece, where the disease has been detected and represents an emerging threat to chestnut production.

Curvularia americana and Curvularia tropicalis cause leaf and crown necrosis on Bermuda grass in Italy

Curvularia americana and C. tropicalis are described as causes of leaf and crown necroses on Bermuda grass (Cynodon dactylon x Cynodon transvaalensis) in Veneto, Northern Italy. These pathogens were characterized using morphological characters, and a multilocus molecular phylogenetic analysis based on the nuclear ribosomal internal transcribed spacer (ITS) region, the partial glyceraldehyde-3-phosphate dehydrogenase (gapdh) and translation elongation factor-1α (tef1-α) genes. Pathogenicity tests and the fulfilment of Koch’s postulates confirmed C. americana and C. tropicalis as foliar pathogens of Bermuda grass. This is the first report of C. americana and C. tropicalis as pathogens of Bermuda grass, and the first record these two fungi in Europe.