Β-tubulin Gene Sequences Research Articles

Identity, Pathogenicity, and Genetic Diversity of Lasiodiplodia Species Associated with Stem-End Rot of Avocado in China.

Stem-end rot (SER) causes brown necrotic lesions in the pulp near the base of the fruit pedicel and is one of the most devastating postharvest diseases of avocados in all avocado-growing regions of the world. China's avocado industry is growing very rapidly, and the planting area is expanding, but little is known about the pathogens and genetic diversity of avocado SER. To determine the causal agents of SER, avocado fruits were sampled from the main avocado-producing areas in China during 2020 and 2021. Fungal isolates were obtained from SER symptomatic avocado fruits and identified by morphology combined with phylogenetic analysis of internal transcribed spacer (ITS), translation elongation factor 1-α (EF1-α), and β-tubulin (TUB2) gene sequences. All 101 isolates belonged to Lasiodiplodia spp.; four Lasiodiplodia species were identified, namely, L. pseudotheobromae (59.41%), L. theobromae (24.75%), L. mahajangana (7.92%), and L. euphorbiaceicola (1.98%); and six others are classified as Lasiodiplodia sp. (5.94%). There were only slight morphological differences in colonies and conidia of these four species of Lasiodiplodia. The pathogenicity tests showed symptoms of SER, and 92.08% of the isolates exhibited a high level of virulence on avocado (disease index >70), related to the disease severity on avocado fruits. All tested isolates grew well under the temperature ranging from 23 to 33°C. There was a significant difference in mycelial growth between the four species of Lasiodiplodia after treatment with high or low temperatures. The growth of L. pseudotheobromae was the fastest at 13 to 18°C but was the lowest at 38°C (P < 0.05). The red pigment could be produced by all tested isolates after culturing for 7 days at 38°C. The mycelial growth rate was the fastest on PDA medium, and the slowest on the OMA medium but promoted spore formation (P < 0.05). In addition, the genetic diversity of pathogenic Lasiodiplodia species associated with SER collected from avocado, mango, guava, and soursop fruits was determined. A total of 74 isolates were clustered into four main ISSR groups by the unweighted pair-group method with arithmetic mean analysis, and the classification of this group was related to the host. Extensive diversity was detected in the Lasiodiplodia populations. The diverse geographical origins and host species significantly influenced the population differentiation, and most of the genetic variation occurred within populations (P < 0.001). This is the first study to identify the major pathogens of avocado SER in China, survey their occurrence and pathogenicity, and include a comparative analysis of genetic diversity with Lasiodiplodia spp. causing SER on other fruit hosts. Collectively, the Lasiodiplodia species complex affecting avocado showed high pathogenicity and diversity, while L. pseudotheobromae was the most frequently isolated species in China. The results of this study provide insights into the aspects of the epidemic of SER disease caused by Lasiodiplodia species, which will help in developing strategies for the management and control of SER in avocado.

Anthracnose of Macleaya cordata Caused by Colletotrichum aenigma in China.

Macleaya cordata (Willd.) R. Br. is a perennial herbaceous medicinal plant (Papaveraceae) commercially cultivated in China which has been studied for detumescence, detoxification, and insecticidal effect (Lin et al. 2018). In August 2021, anthracnose was observed in 2-year-old M. cordata plants in Benxi county, northeast China (41°45'48″N, 123°69'15″E). Dozens of irregular reddish-brown spots (3-11 mm) were observed on each diseased leaf. The lesions were covered with a layer of gray-white mycelia. As the disease progressed, the spots became necrosis and perforation or they would merged into large lesions, ultimately resulting in wilted leaves (Fig. 1). More than 33% of the plants in a 16-ha field were infected in 2021. The diseased leaves were collected and cut into 3-8 mm pieces, surface-disinfested by immersing them into 1% NaOCl for 2 min, and rinsed three times with sterile distilled water. They were then dried with sterilized absorbent paper, placed on PDA medium amended with chloramphenicol (40 mg/L), and incubated in darkness at 25°C with a 12-h photoperiod. Twenty isolates (BLH1 to 20) were obtained and purified using a single-spore method. Isolate BLH12 was identified and used for the pathogenicity test. Colonies were sparsely fluffy with smooth edges, and gradually became gray to pale orange from the initial white. The underside of the colonies was pale orange towards the center. Conidia were single-celled, cylindrical, and transparent with broadly blunt ends, measuring (15.13 ± 1.14) × (5.80 ± 0.60) μm (n=50). Appressoria were single-celled, brown-to-dark brown, usually elliptical or irregular, and sometimes lobed. Setae were not observed. The isolate was initially identified as Colletotrichum gloeosporioides complex (Prihastuti et al. 2009). The identification was confirmed as described previously (Weir et al. 2012). The rDNA internal transcribed spacer region (OP415560), the glyceraldehyde-3-phosphate dehydrogenase (OP433642), chitin synthase (OP433643), calmodulin (OP433644), actin (OP433645), glutamine synthetase (OP433646), β-tubulin (OP433647), and superoxide dismutase (OP433648) gene sequences were obtained (Carbone & Kohn 1999; Weir et al. 2012), and BLAST searches revealed 99-100% homology with the type culture ICMP 18608 (JX010244, JX010044, JX009683, JX009443, JX009744, JX010078, JX010389, and JX010311). A phylogenetic analysis of combining all loci indicated BLH12 and the type strain of C. aenigma were clustered in one group (Fig. 2). Based on the basis of morphological characteristics and phylogenetic relationships, BLH12 was identified as C. aenigma. For the pathogenicity test, healthy 2-year-old plants were sprayed with a BLH12 spore suspension (1 × 105/mL). Control plants were sprayed with sterile water.There were three replicates (five plants each) per treatment. All plants were incubated at 25°C (12-h photoperiod and 86% relative humidity) and examined after 7 days. The experiment was repeated twice. The inoculated plants showed lesions on the leaf surface, similar to those in the field, whereas the control plants were asymptomatic. The pathogen was successfully reisolated and identified as the methods mentioned above. This fungus reportedly infects the leaves of many woody plants in China (Wang et al. 2020; Zhang et al. 2021). This is the first report of C. aenigma causing anthracnose on M. cordata, which will provide an guideline for developing effective field control practices for the disease.

Nigrospora musae and N. oryzae as new causal agents of broad bean leaf spot disease in China

Broad bean (Vicia faba L.) is a cold-season legume crop, planted worldwide for its superior nutritional value and important role in sustainable agriculture. Leaf spot is one of the major diseases on broad bean leaves, stems and pods, the infections will result in severe yield reduction. In 2021, a leaf spot disease was extensively observed on broad bean plants in Longli County and Guiyang, Zunyi and Liupanshui Cities, Guizhou Province, China. Based on morphology and phylogenetic analysis using the ribosomal internal transcribed spacer region (ITS), 28S rRNA gene (LSU), β-tubulin (TUB2) and translation elongation factor 1-alpha (TEF1-α) gene sequences, the 12 isolates in this study represent two species of Nigrospora, namely N. musae, and N. oryzae. Moreover, the isolates of the two Nigrospora species found in the survey were tested for pathogenicity and indicated that they were all pathogenic to the broad bean plants. Same pathogens were successfully reisolated from the typical leaf spot lesions. Koch's postulates for N. musae and N. oryzae were fulfilled in this study. Thus, this study confirms species of Nigrospora as causal agents of leaf spot disease on broad bean in China, which provides a basis for subsequent research to develop effective preventative measures.

Neofusicoccum parvum: A NOVEL PATHOGEN SPECIES CAUSING WILTED LEAF AND DIEBACK PETIOLES ON LOTUS (Nelumbo nucifera) IN THUA THIEN HUE, VIETNAM

Lotus (Nelumbo nucifera) is an essential species in many countries. In Vietnam, the lotus is a plant with cultural and spiritual significance, representing purity, spiritual growth, and enlightenment. However, petiole dieback and wilted dry leaves are severe diseases that weaken the host and decrease leaf photosynthesis, reducing lotus production. In this study, a new dieback and wilted leaf pathogen were identified via its morphology, phylogeny, and pathogenicity. Four representative Botryosphaeriaceae isolates from lotus fields in Hue, Phong Dien, and Phu Loc were selected for identification and pathogenicity testing. Based on morphological and phylogenetic analyses and by using the ribosomal internal transcribed spacer region (ITS) and β-tubulin (tub-2) gene sequences, we identified four isolates as Neofusicoccum parvum. In the pathogenicity test, typical symptoms appear on the inoculated lotus petioles, including dieback, curving, and wilted leaves. These symptoms are consistent with those observed in the field. In addition to identifying the pathogen species responsible for lotus disease, this study provides valuable insights into the taxonomy and phylogenetic relationships of new fungal pathogens that affect lotus fields. These findings can contribute to effective management strategies to control these diseases and improve lotus production. To the best of our knowledge, this is the first report of characterization and phylogenetic analysis of N. fusicoccum as the causal agent of wilted leaves and dieback disease in Thua Thien Hue, Vietnam.

First report of Leptosphaeria biglobosa 'brassicae' causing leaf spots in Eutrema japonicum in Yunnan, China.

Wasabi (Eutrema japonicum) is a root vegetable that is cultivated at large scales in southwestern China. In November 2021, approximately 40% of plants in a forested plantation in Dadishui, Yunnan Province, China (25.47°N, 103.22°E), showed leaf spot symptoms. The early symptoms were small black spots that gradually expanded into irregular brown to black lesions (0.5-1.5 cm), which were restricted by leaf veins. Yellow halos were observed at the outer edges of necrotic lesions. To identify the causal agent, we collected 20 diseased leaves and obtained fungal isolates from symptomatic leaf tissues. Following surface sterilization with 75% ethanol for 30 s, the tissues were cultured on potato dextrose agar (PDA) plates and incubated at 25°C under a 12 h light/12 h dark light cycle. After 7 days of incubation, a total of 12 isolates were obtained through single-spore culture. All isolates had similar colony morphology, and produced fluffy white mycelia and yellow pigment after 1 week of PDA culture at 25°C, and blackish- brown mycelium, tan pigment, and conidia after 2 weeks. The conidia were hyaline and cylindrical, with an average size of 4.6 μm × 2.2 μm. These morphological characteristics similar to the description of Leptosphaeria biglobosa (Shoemaker et. al, 2001) and Leptosphaeria maculans (Vincenot et al. 2008). Genomic DNA was extracted from mycelium of isolate SK-1, which was harvested from 10-day-old PDA culture using a FAST plant genomic DNA Extraction Kit (Biomed, China), following the manufacturer's instructions. The species-specific primers LbigF, LmacF, and LmacR (Liu et al. 2006) were used for identification via polymerase chain reaction (PCR). A 444-bp fragment characteristic of L. biglobosa 'brassicae' (Lbb), and a 330-bp of L. maculans 'brassicae' (Lmb) were amplified, respectively. Internal transcribed spacer (ITS) sequences (592 bp), part of the 5' end of beta-tubulin (968 bp), and actin (899 bp) were also amplified using the primers ITS1/ITS4, BT1/BT2, and ACTF/ACTR (Vincenot et al. 2008), respectively. PCR was performed in a volume of 25 μL containing 12.5 μL 2 × T5 Super PCR Mix (Tsingke Biotech, Beijing, China), 1 μL 10 μM primer (Tsingke Biotech), 1 μL DNA template, and an aliquot of sterile water to attain the total volume. The thermal cycler settings were 5 min at 98°C; 35 cycles of 10 s at 98°C, 10 s at 58°C, and 30 s at 72°C; and extension for 2 min at 72°C. The ITS sequence of isolate SK-1 (GenBank accession no. OQ216838), the partial β-tubulin gene sequence (OQ241183), and the actin gene sequence (OQ241184) indicated 100% query cover and 100% identity with L. biglobosa (DQ458906), Lbb strain B3.6 (AY748995), and Lbb strain 2379-4 (AY748949), respectively. Phylogenetic analysis (King et al. 2022) also identified of isolate SK-1 as Lbb. To determinate its pathogenicity, isolate SK-1 was grown on PDA incubated at 28°C for 2 weeks, and conidial suspensions were prepared at a concentration of 106 conidia/mL. Then, 15 leaves of 4-month-old E. japonicum seedlings were needle-wounded on the front and inoculated by syringe injection of 10 μL of the appropriate conidial suspension. We used 10 μL of the sterilized distilled water as the control under forest growth conditions. All inoculation sites were covered with cotton strips and moistened with 1.0 mL sterile water to maintain humidity. After 12 days of incubation, the leaves developed symptoms similar to those observed in the field, and the fungus was reisolated from diseased leaves, whereas the controls remained healthy. Based on these results, we identified L. biglobosa 'brassicae' as the causal agent of leaf spot on E. japonicum in China. This fungus has been reported to cause blackleg in many Brassica crops in China such as Brassica napus (Fitt et al. 2006), Brassica oleracea (Zhou et al. 2019), B. juncea var. tumida (Deng et al. 2020), Brassica rapa subsp. pekinensis (Yu et al. 2021). To the best of our knowledge, this is the first report of L. biglobosa causing leaf spots in E. japonicum in China. Our data provide a basis for disease management in E. japonicum production in China.

Diversity Profiling and the Distribution of Pathotypes and Mating Types in Indian Isolates of Ascochyta rabiei Causing Ascochyta Blight of Chickpea

The prevalence and distribution of pathotypes and mating types of Ascochyta rabiei (Pass.) Labr. were evaluated during the winter (October to March) seasons of 2017-18 and 2018-19. Forty-five A. rabiei isolates were obtained from six different states. From the survey results, the state of Punjab recorded the highest disease rating (Rating 9) compared with other chickpea-growing areas. The β-tubulin gene was amplified (330 bp) from all the isolates, and sequence analysis was performed. The phylogenetic tree produced by cluster analysis of β-tubulin gene sequences differentiated the 44 A. rabiei isolates . Using multiplex PCR, mating types were identified. From the mating type (MAT) study of all the A. rabiei isolates, amplification was observed at the 490-bp size, which corresponds to mating type 2 (MAT1-2). The four pathotypes (I, II, III, IV) and six physiological races were delineated based on the differential reaction of the disease. The most widely distributed pathotype was pathotype III (52.27%) among the surveyed regions of North Indian states, except Uttarakhand (where only pathotype II was recorded), followed by the least aggressive pathotype I (22.7%), highly aggressive pathotype IV (15.9%), and aggressive pathotype II (9.1%). Among the six physiological races identified, the most common races were race 5 (25%), followed by race 1 (22.73%), race 6 (18.18%), race 4 (18.18%), race 3 (9.09%), and race 2 (6.82%). Because of the prevalence of a single mating type among the surveyed locations in India, we opine that there may be less diversity in A. rabiei compared with isolates reported from other countries. However, to substantiate this conclusion, evaluation of more isolates of A. rabiei is required. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Emergence of Ancylostoma caninum parasites with the benzimidazole resistance F167Y polymorphism in the US dog population

BackgroundAnthelmintic resistance to benzimidazole has been detected in the canine hookworm, Ancylostoma caninum. Benzimidazole resistance is believed to have developed originally in greyhounds, but has also been detected in non-greyhound pet dogs. The aim of this study was to validate a probe-based allele-specific real-time PCR tests for the F167Y polymorphism on the β-tubulin isotype-1 gene and to determine the geographic distribution. MethodsAllele-specific real-time PCR tests were established and validated to detect the codon 167 polymorphism in the Ancylostoma caninum β-tubulin isotype-1gene. Additionally, real-time PCR tests were validated for Ancylostoma spp. and Uncinaria stenocephala. Two nucleic acid extraction protocols were validated including mechanical disruption of parasite structures in stool. The frequency of the F167Y single nucleotide polymorphism (SNP) was determined in hookworm confirmed stool samples. Samples with the resistant 167Y genotype were confirmed by β-tubulin gene sequencing and allele frequencies were determined. ResultsThe Ancylostoma spp. and A. caninum F167Y allele-specific real-time PCR tests were highly sensitive and specific when tested against synthetic DNA, spiked samples, and characterized parasites. Using an optimized total nucleic acid extraction protocol, 54 of 511 (10.6%) were found to contain the benzimidazole resistance allele. All 55 samples containing hookworms with the resistance mutation were confirmed by β-tubulin gene sequencing. The majority of resistant hookworms (44 resistant, 183 tested; 24.4%) originated from Florida, five from California (103 tested, 4.9%), three from Idaho (40 tested, 7.5%), two from Nevada (22 tested, 9.1%), and one sample from Hawaii (13 tested, 7.7%). Resistant genotypes were found in 14 different dog breeds including eight in Greyhounds. Allele-frequency determination revealed resistance allele frequencies between 1 and 100% with 58% above 50%. ConclusionsThis data strongly supports recent findings of benzimidazole resistant canine hookworms present throughout the general US pet dog population.

First Report of Neopestalotiopsis Disease in Ohio caused by an emerging and novel species of Neopestalotiopsis on Strawberry.

In October 2021, strawberry (Fragaria x ananassa) plants (cv. Ruby June) that had dark brown lesions with a diffuse black halo and light brown center and / or dark brown V-shaped necrotic areas often starting from the edge of the leaves were observed in a commercial planting in Washington County. The grower reported 50% incidence in the field when the sample was first submitted and two weeks later reported 80% incidence. The morphology of conidia present on symptomatic leaf tissue was consistent with species of Neopestalotiopsis (Maharachchikumbura et al. 2014). The conidia were ellipsoid to fusiform, five-celled, with three light brown colored median cells and one hyaline apical and basal cell. The apical cells had two to four flexuous appendages and the basal cell had one non-flexuous appendage. Average (N=30) conidia length, not including the appendages, and width was 24.1 ± 2.7 and 6.5 ± 1.4 µm respectively. Two isolates (MLI267-21 and MLI268-21) were purified on potato dextrose agar, producing a dense white mycelial mat with undulate margins. The underside color of the mycelial mat was pinkish-orange. Conidiomata formed randomly in the colonies and extruded black gelatinous spores. To confirm the identity of these isolates the genome of MLI267-21 was sequenced using the NextSeq 2000 Illumina platform and Nextera DNA CD indexes (OSU Applied Microbiology Service Laboratory, Columbus, OH). Partial internal transcribed spacer (ITS) region, β-tubulin (TUB), and translation elongation factor 1-alpha (TEF-1α) gene sequences (Accession numbers: OM649904, OM649905, and OM649906 respectively) were extracted from the MLI267-21 genome, concatenated, and aligned to published reference sequences. These same genes were amplified and sequenced from MLI268-21. Maximum likelihood phylogenetic analysis performed in IQ-TREE (Minh et al. 2020, Kalyaanamoorthy et al. 2017, Chernomor et al. 2016) with the aligned sequences revealed the clustering of MLI267-21 and MLI268-21 with seven other Neopestalotiopsis isolates, from strawberry (17-43L; Baggio et al. 2021) and pomegranate (GEV3426 to GEV3431; Xavier et al. 2021) leaves in Florida, which form a unique and emerging species group. The ITS, TUB, and TEF-1α sequences from both Ohio isolates were 100% similar to the same sequences from 17-43L and GEV3426 - GEV3431. Pathogenicity tests were performed using MLI267-21 by spray inoculating (~104 spore/ml) four-week-old 'Cabrillo' strawberry plants (n=4) and placing three drops (10µl each) of spore suspension (~104 spore/ml) on the calix area of detached fruit (n=4). Non-inoculated plants and fruit (n= 4 each) served as negative controls. The plants were covered with transparent plastic bags and maintained at 25 °C for 72 hours before the bags were removed (Baggio et al. 2021). Five days post-inoculation, dark brown circular spots on the leaves and petioles were observed on all four inoculated plants and acervuli were observed within the necrotic spots after an additional 72 hours in a moist chamber. Fruits were incubated in a moist chamber at 25 °C and after 72 hours orange-brown lesions formed on the fruit. After five days, fruit were mushy and covered with white mycelia, acervuli, and conidiomata. Neopestalotiopsis disease has been reported on strawberry in Florida (Baggio et al. 2021) and in several South American (Obregon et al. 2018, Hidrobo et al. 2021) and European (Chamorro et al. 2016, Gilardi et al. 2019) countries. The disease can cause rapid plant death when conditions are warm and wet. Research to investigate host susceptibility and to identify effective chemical and biological control has been initiated in Ohio to establish preventative management programs for commercial field operations.

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.

Clinical and Microbiological Characteristics of Aspergillosis at a Chinese Tertiary Teaching Hospital.

Aspergillus spp. infection in immunocompromised patients results in increasing morbidity and mortality. This study investigated clinical and microbiological characteristics of aspergillosis in a Chinese tertiary teaching hospital. A total of 114 patients with aspergillosis were included over a 5-year period at Ruijin Hospital. In sum, 114 Aspergillus strains were isolated and identified at species level using matrix-assisted laser desorption ionization time-of-flight mass spectrometry, confirmed by ITS gene region and β-tubulin (BenA) gene sequencing. Sensititre YeastOne was used in vitro to test susceptibility to antifungal drugs: amphotericin B, itraconazole, voriconazole, posaconazole, isavuconazole, micafungin, anidulafungin, and caspofungin. The median age of the patients was 61 (19) years, men accounted for 53.5% (n=61) of the sample, about 64% were immunocompromised, and 36% had underlying diseases. Pulmonary diseases accounted for 27.2%. Aspergillus isolates were mainly isolated from sputum (n=42, 36.8%). Antifungal therapy was administered to 106 (93.0%) patients and voriconazole (n=76, 66.7%) was the most frequently used as empirical therapy. Aspergillus fumigatus (n=69, 60.5%) was the most common species. There was a 73.7% concordance between MALDI-TOF MS and molecular identification. All Aspergillus isolates showed good susceptibility to anidulafungin and caspofungin. Immunocompromised patients are an at-risk population for aspergillosis, and voriconazole was used as empirical therapy in Ruijin Hospital, China. A. fumigatus was the predominant Aspergillus species causing aspergillosis, and A. flavus - as non-A. fumigatus species are increasing - the second-leading cause of aspergillosis. Anidulafungin and caspofungin were the most active in vitro against the Aspergillus isolates tested. The MALDI-TOF MS method showed good accuracy for identification of common Aspergillus spp. In vitro antifungal-susceptibility testing is crucially important for decisions on effective therapy with aspergillosis.

A SYBR Green qPCR assay for specific detection of Colletotrichum ocimi, which causes black spot of basil

Colletotrichum ocimi causes black spot of basil (Ocimum basilicum) and is a serious threat to basil cultivation as it compromises leaf production. The pathogen also infects seeds, which could become primary sources of inoculum for spread of black spot. A SYBR Green real-time PCR assay was developed to detect Colletotrichum ocimi in basil leaves and seeds, based on the partial β-tubulin (tub2) gene sequence. Two primer sets were designed and tested. The selected primer pairs produced amplicons of 130 bp. The real-time PCR assay was validated for analytical specificity, sensitivity, selectivity, repeatability and reproducibility. The assay was specific for C. ocimi with respect to ten Colletotrichum spp. and to another 12 pathogens of basil plants. Sensitivity was 1 pg µL-1 of genomic fungal DNA and amplification analyses were not influenced by basil genomic DNA. The assay detected and quantified C. ocimi in artificially inoculated basil leaves. This is the first specific primer set for C. ocimi, which allows rapid detection and quantification of the pathogen is a useful tool for diagnostics in plants. Detection in seeds would also be possible, but will require an optimized extraction method. The qPCR detection of C. ocimi in planta can contribute to adoption of effective preventive disease management strategies.

Molecular and Morphological Characterization of Exserohilum turcicum (Passerini) Leonard and Suggs Causing Northern Corn Leaf Blight of Maize in Bihar.

Maize is considered the third most important cereal crop in Asia after rice and wheat. Many diseases affect this crop due to the cultivation of various hybrids. This research aimed to characterize the causative agent of northern corn leaf blight disease in Bihar, India, caused by Exserohilum turcicum (Passerini) Leonard and Suggs. Leaf samples were collected from infected fields in five maize growing districts of Bihar in 2020–2022. A total of 45 fungal isolates from 135 samples were examined for cultural, morphological, and molecular characteristics and were identified as E. turcicum. The isolates were grouped into four groups based on colony color, i.e., olivaceous brown, blackish brown, whitish black, and grayish, and into two groups based on regular and irregular margins. The conidial shapes were observed to be elongated and spindle-shaped with protruding hilum, with conidial septa ranging from 2–12. Similarly, conidial length varied from 52.94 μm to 144.12 μm. β-tubulin gene sequences analysis made it possible to verify the identities of fungal strains and the phylogenetic relationships of all isolates, which were clustered in the same clade. The β-tubulin gene sequences of all the isolates showed a high level of similarity (100%) with reference isolates from GenBank accession numbers KU670342.1, KU670344.1, KU670343.1, KU670341.1, and KU670340.1. The findings of this study will serve as a baseline for future studies and will help to minimize yield losses.

Enzyme activity of Aspergillus section Nigri strains isolated from the Korean fermentation starter, nuruk.

Aspergillus section Nigri is a fungus used industrially because of its ability to produce enzymes such as cellulolytic, amylolytic and proteolytic enzymes. In this study, we obtained twenty-eight strains of Aspergillus section Nigri from the traditional Korean fermentation starter, nuruk, which is known as a mixed culture of enzymatic filamentous fungi and yeasts. All strains were identified as Aspergillus section Nigri through combined phylogenetic analysis using partial β-tubulin and calmodulin gene sequences. The cellulase, amylase and protease activities of Korean strains were measured and compared with ten reference strains of Aspergillus niger. Most Korean strains showed higher cellulolytic activity than reference strains, and Aspergillus neoniger KCN5 showed the highest β-glucosidase activity. Two-thirds of the Korean strains showed similar levels of α- and glucoamylase activity as the reference strains. The protease activity of Aspergillus section Nigri strains was the highest at pH 3.0, and A. niger KSJ2 showed the highest acidic protease activity. By comparing ten reference strains and twenty-eight Korean strains, our results suggested useful Aspergillus section Nigri strains from nuruk with high enzyme activity, such as KCN5 and KSJ2, and their potential for industrial applications as enzyme producers.

First report of Fusarium wilt caused by species of the Fusarium solani complex on sweet cherry in Henan province, China

Sweet cherry (Prunus avium) is an important fruit crop in China with a cultivation area of more than 13,000 km2 (Sun et al., 2017). In June 2020, wilt symptoms caused by unknown factors occurred on four-year-old sweet cherry (cv. Gisela 5) with 30% disease incidence in an orchard of Nanyang Agricultural Vocational and Technical College (32°59´5˝N, 112°30´38˝E), Henan, China. Affected leaves were predominantly curved at first (Fig. 1a), and withered under intense sunlight and drought. The root surfaces were dark black (Fig. 1b). The cross-sections of affected roots were brown with the bark rotted (Fig. 1c). Affected root segments were rinsed with water and treated with 75% (v/v) ethanol for 15 s, followed by 4 min in 0.1% (v/v) HgCl2 then rinsed five times with sterile distilled water. Internal diseased tissue pieces were cut out with a sterilised knife and placed onto potato dextrose agar (PDA) plates. Two isolates FS-1 and FS-2 were selected randomly from 14 Fusarium-like isolates growing from the symptomatic tissues. The isolates produced off-white colonies (Fig. 2) and three types of asexual spores, i.e. microconidia, macroconidia and chlamydospores on PDA after five days of incubation at 28°C (Fig. 3). Microconidia were hyaline, oval or elliptical in shape with 0–1 septum and measured 8.2 to 19.6 (12.7) μm × 3.2 to 5.3 (4.1) μm (n = 50) in size. Macroconidia were hyaline, mostly crescent-shaped with 2–5 (3) septa and measured 26.4 to 41.1 (34.6) μm × 3.8 to 6.4 (5.0) μm (n = 50). Chlamydospores were brown, round in shape with a diameter of 5.8-10.8 (8.0) μm (n = 100), frequently born on a conidium or between two hyphal cells. Microconidia were produced commonly prior to macroconidia and chlamydospores. The morphological features of the isolates agreed with those of the F. solani species complex that consisted of multiple Fusarium spp. strains including the members previously belonged to F. solani (Matuo and Snyder, 1973). Sequences of rDNA-ITS (internal transcribed spacer), tef (translation elongation factor 1 α gene) and β-tubulin gene of the two isolates were amplified with primers ITS1/ITS4, EF1-688F/EF1-1251R and Bt2a/Bt2b, respectively (Keeling, 2003; Lu et al., 2011). The GenBank Accession Nos. were MW276145 and MW276147 for rDNA-ITS, MW303522 and MW303523 for tef, and MW303520 and MW303521 for β-tubulin gene of FS-1 and FS-2, respectively, with a maximal sequence identity of 99.82-100% to multiple F. solani species complex strains. In the neighbour joining phylogenetic trees of amplified sequences, both FS-1 and FS-2 clustered with F. solani species complex strains, clearly separating them from other Fusarium spp. (Fig. 4). For the pathogenicity tests, the isolates were grown on PDA at 28°C for five days. Mycelial plugs (5 mm diameter) were inoculated on wounds made 10 cm above the ground of two-year-old healthy cv. Gisela 5 stalks (n = 3 for each isolate). The inoculated sites were wrapped with plastic tape. For the controls (n = 3), the wounds were inoculated with PDA plugs alone. After 30 days incubation at 24°C and 65 % RH, the plants inoculated with FS-1 and FS-2 showed wilt symptoms similar to those observed on naturally affected plants. The control plants were asymptomatic (Fig. 5). Fusarium cultures with the same phenotypic features as the inocula were reisolated from diseased tissues of the inoculated plants, confirming FS-1 and FS-2 as causal agents of the disease. F. ventricosa (Chen et al. 2016) and F. oxysporum (Úrbez-Torres et al. 2016) were reported as pathogens causing root rot on sweet cherry in China and British Columbia, respectively. This is the first report of Fusarium wilt caused by F. solani species complex on sweet cherry in China.

First report of cauliflower anthracnose caused by Colletotrichum gigasporum in India

Cauliflower (Brassica oleracea var. botrytis) is an important vegetable in India, where it is mostly grown in the states of Bihar, Gujarat, Haryana, Madhya Pradesh and West Bengal. India is the world's second largest producer of cauliflower with over 8.84 million tonnes produced annually (Food and Agriculture Organization, 2020). In August 2019, sunken, dark brown stem lesions, covered with acervuli, were observed on cauliflower seedlings in commercial fields in Jalna, Maharashtra, India (Figure 1). Approximately 5% of the seedlings were affected by the disease. Five diseased cauliflower seedlings were collected, and surface sterilised with 1% NaOCl for two minutes followed by two washings with sterile distilled water. Surface-sterilised sunken necrotic lesions on the stems were examined under the microscope. Conidial suspensions were obtained by dispensing 20 µl of distilled water onto acervuli. Serial dilutions of the suspensions were plated onto potato dextrose agar (PDA) with 50 mg/l streptomycin sulphate to obtain pure cultures. The plates were then incubated at 28°C with a 16 h light/8 h dark cycle for seven days. The fungal colonies were grey with scattered black conidial masses (Figure 2). The mycelial growth rate on PDA was 10 mm per day. Conidia were cylindrical, thin walled, hyaline and aseptate. The conidia measured 21.1 ± 1.0 × 8.3 ± 0.4 µm (Figure 3). These morphological characters align with the description of the Colletotrichum gigasporum species complex (Liu et al., 2014). For molecular characterisation, the glyceraldehyde 3-phosphate dehydrogenase (GAPDH), parts of the β-tubulin and actin (ACT) genes were amplified (Liu et al., 2014) and sequenced. BLAST analysis in the NCBI database showed 100% identity to GAPDH, β-tubulin and ACT gene sequences of C. gigasporum (GenBank Accession Nos. KF687829, KF687872 and KF687787, respectively). The sequences were deposited in GenBank (MW307265-MW307267) and aligned with published sequences using MEGA11 (Tamura et al., 2021), followed by phylogenetic analysis using the UPGMA method (Figure 4). Phylogenetic analyses confirmed the identification as C. gigasporum. To confirm the pathogenicity of the isolates, leaves of five potted 40-day-old cauliflower seedlings were surface sterilised with 70% ethanol for 30 seconds and inoculated with 10 µl of conidial suspension (c. 105 conidia/ml) obtained from a seven-day-old culture grown on PDA. Leaves of an additional five seedlings were inoculated with sterile water as a control. Typical anthracnose symptoms developed on C. gigasporum-inoculated cauliflower seedlings seven days after inoculation (Figure 5) and no symptoms were seen on control plants. The pathogenicity experiment was repeated twice with similar results. Conidia were re-isolated from symptomatic cauliflower seedlings. Colony morphology and conidial characteristics were identical to the cultures used for inoculation, thereby fulfilling Koch's postulates. Colletotrichum gigasporum has been reported as a causal agent of anthracnose of avocado (Persica americana) in Sri Lanka (Hunupolagama et al., 2015) and of fragrant rosewood (Dalbergia odorifera) in China (Wan et al., 2018). Colletotrichum gigasporum has not been reported previously in India. To our knowledge, this is also the first report of anthracnose on cauliflower caused by C. gigasporum. The authors are thankful to Mahyco Research Centre, Maharashtra for support and suggestions.

First Report of Root Rot Disease in Green Gram (Vigna radiata) Caused by Ectophoma multirostrata in India.

A disease survey was conducted in the green gram (Vigna radiata) growing areas of Bharuch, Gujarat, India in February 2020. Root rot was observed on cultivar NVL-585 in 30 fields surveyed, with 15 to 20% of disease incidence. Infected plants showed foliar chlorosis, brown discoloration, dark black brittle roots with necrotic lesions, and dead plants. The root rot causal agent was isolated on potato dextrose agar (PDA) from 25 symptomatic samples collected from 10 diseased plants and pure cultures were obtained using single hyphal tip method. The cultures were grayish black with a smooth texture. Conidia were single celled, elliptical to oval in shape and were 5.2 to 6.5μm (n=25) in length and 2.7 to 3.4µm in width. Plastic pots (15 cm in diameter) were filled with autoclaved soil (1.5 kg/pot) inoculated with a 15-day-old culture of the fungus at 3 g/ kg (108 cfu/g). After 7 days, the local green gram cultivar HUM 16 was sown into the pots. Seedlings were thinned to 10 for each pot. Chlorosis of leaves, rotting roots, and even plant death were observed at 20, 30 and 40 days after seeding. The experiment was conducted twice to fulfill the Koch postulates. The fungus was reisolated from the root rot samples, and identical colony morphology was observed as seen previously. Based on the cultural characteristics and conidial morphology, the fungus belonged to Ectophoma sp. (Boerema et al. 2004). ITS, D1 and D2 regions of LSU and β-tubulin were used for molecular identification. The BLASTn analysis of ITS, D1 and D2 region of LSU gene and β-tubulin gene sequences of Isolate G80 showed a 100% similarity with accession number MH858623 (Vu et al. 2019), 99.82% similarity with MH870612 (Vu et al. 2019) and 100% similarity with MN983939 (Hou et al. 2020). Gene sequence has been submitted to NCBI GenBank, and accession numbers for ITS, D1 and D2 region of LSU and β-tubulin are MW165415, MW813868 and LC656357, respectively. The pathogen was previously reported from other hosts like Fuchsia× hybrida, Origanum vulgare, Lavandula angustifolia, Cicer arietinum and Coriandrum sativum. It can cause substantial economic damage to a wide range of commercially important crops. To the best of our knowledge, this is the first report of E. multirostrata causing root rot disease of green gram in India.

First Report of Pulp Rot in Externally Asymptomatic Pomegranate Fruit Caused by Talaromyces albobiverticillius in Henan Province, China.

As a popular deciduous fruit tree, pomegranate (Punica granatum L.) is grown from tropical to temperate zones worldwide, therein China has at least 120000 hm2 cultivation area. In August 2020, severe pulp rot occurred in the externally asymptomatic pre-harvest pomegranate fruit on a 3-year-old soft-seeded variety (Tunisia) in the Zhanghe village (32º40´34˝N, 111º44´20˝E) of Jiuchong township, Xichuan county in Henan province, China with 6.4-20 (av. 12.6) % pulp rot incidence evaluated from 11 freshly sampled fruits (360 pulps per fruit investigated). The fruits showed no external symptoms, however, browning occurred on part of their pulps before harvest compared to the normal ones with white or pink color. The surface of the externally asymptomatic fruits was sterilized with 75% ethanol, and air-dried in a clean bench. The surface-disinfected fruits were dissected with a sterilized knife. Brown pulps from the fruits were picked up using flame-sterilized tweezers and placed on potato dextrose agar (PDA) plates. After five days of incubation at 28 °C, pure fungal cultures with similar phenotypic features developed from the affected pulps. Two randomly selected isolates Tp-2 and Tp-8 were used for the study. The colony surface of the isolates was greyish-green with claret-red exudates. Claret-red pigments were commonly secreted into the medium from the colonies. Conidia were unicellular, hyaline to greyish, mostly rugby ball-shaped with a dimension of 2.2-3.5 (2.7) µm × 1.6-2.0 (1.8) µm (n=50) for Tp-2, and 2.2-3.1 (2.6) µm × 1.6-2.2 (1.8) µm (n=50) for Tp-8. The rDNA internal transcribed spacer (ITS) and β-tubulin gene sequences of the isolates were amplified with primers ITS1/ITS4 and Bt2a/Bt2b, respectively. Sequences were submitted to GenBank with accession numbers MW132153 and MW132077 for the rDNA-ITS sequences, and MW507822 and MW507823 for the β-tubulin gene sequences of Tp-2 and Tp-8, respectively, with a maximal sequence identity greater than 99 % to multiple strains of Talaromyces albobiverticillius (TA) based on BLAST analyses. In the Neighbor-joining phylogenetic trees constructed using rDNA-ITS and β-tubulin gene sequences, both Tp-2 and Tp-8 formed a clade with mutiple strains of TA, clearly separated from other Talaromyces spp. Conidial suspensions (106 spores ml-1) of Tp-2 and Tp-8 were separately injected into five pomegranate fruits (Tunisia) sampled from an orchard free of the disease with a sterilized syringe. Five fruits inoculated with sterilized water were used as control (CK). The inoculated fruits were incubated at 25 °C for 10 days and cut out through the inoculated sites. Pulp rot symptoms occurred in the Tp-2/Tp-8-inoculated fruits, being similar to the naturally affected pulps. The CK pulps remained symptomless during the inoculation tests. Fungal cultures with the same phenotypic features as the inocula were constantly isolated from the brown pulps of the inoculated fruits, verifying both Tp-2 and Tp-8 as the causal agents of the disease based on Koch's postulates. During a long-term (30-40 days) storage at ambient conditions, fruits sampled from affected orchards developed brown lesions on their peels from which TA cultures could be isolated. TA was reported as the pathogen causing postharvest fruit rot on pomegranate in Italy (Mincuzzi et al. 2017). This is the first report of TA causing pulp rot in the externally asymptomatic pomegranate fruit in China.

First Report of Pestalotiopsis microspora Causing Leaf Spot on Moyeam in China.

Ampelopsis grossedentata, commonly known as moyeam, has been widely used as health care herbal tea since it contains natural plant protein cream, 17 amino acids, 14 micronutrients and lots of functional flavonoid and provides a wide range of pharmaceutical functions such as antioxidant, anti-inflammatory, antitumor (Carneiro et al. 2021; Zhang et al. 2020). Moyeam is primarily produced in Zhangjiajie, stretching over the area from between 109'40 to 110'20E to between 28'52 to 29'48N, at 1300 to1890 meter above the sea level, with subtropical humid monsoon climate. Its economic value surpasses $1.25 billion in China (Liang et al. 2020). In July 2020, leaf spots were observed on some moyeam plants in Zhangjiajie. Initial spots were pinhead-sized with a yellow halo margin. The spots developed into light brown necrotic spots 6 to 8 mm in diameter, often with a dark brown margin. After 4 days of development, the spots enlarged and coalesced into irregular shape, frequently falling out and giving the leaves a tattered appearance. The infected plants eventually died with disease incidence ranging from 18 to 23%. This disease resulted in production losses of up to $1.7 million in 2020. One fungal isolate was isolated from the symptomatic leaves based on our previously published methods (Yi et al. 2019). Colonies on potato dextrose agar (PDA) were thick and villous with white at the front of the plate and yellowish at the back. After 1 week, the fungus produced conidia, which were spindle-shaped, straight or slightly curved, with 5 cells, 4-euseptates and 2-3 apical accessory filaments. Morphologically, the fungus was similar to Pestalotiopsis spp. Aerial hyphae with vigorous growth were collected for molecular identification. ITS nucleotide sequence of the rDNA and β-tubulin gene were amplified and sequenced with universal primers ITS1/ITS4 and self-designed primers based on β-tubulin gene conserved motif. BLAST searches against GenBank indicated that the ITS nucleotide sequence shared 99% similarity with that of P. microspora (MG808374.1) and the β-tubulin gene sequence shared 99% similarity with that of P. microspora (AF115396.1). Based on morphological and molecular characteristics, the fungus was identified as P. microspora. ITS and the β-tubulin nucleotide sequences were deposited in GenBank (accession no. MW350011 and MW816914). Pathogenicity tests were carried out with the following procedure. Three healthy moyeam seedlings were sprayed with a conidial suspension of 1 x106 conidia/ml while the other three seedlings were sprayed with distilled water as the controls. Plants were maintained in a greenhouse at 28±1°C. After one day of inoculation, symptoms identical to those in the field developed in the plants inoculated with the fungus. In contrast, no symptoms developed on the control plants. P. microspora has been reported to cause diseases in many crops in China. However, this is the first report of P. microspora causing leaf spot in moyeam in China. Identifying the pathogen causing the disease is important to the development of effective disease management strategies for control of this disease.

Mycobiota of deep-sea benthic communities on the Piip submarine volcano, Bering Sea, Russia

The mycobiota of deep-sea benthic communities on the Piip submarine volcano, Bering Sea, Russia, has been studied for the first time. Filamentous fungi were isolated from deep-sea bottom sediments, sponges, and holothurians using traditional culture-based methods. The fungi were identified using a combination of methods based on morphology of reproductive structures, cultural colony characteristics, and the ITS1-5.8S-ITS2and partial β-tubulin (BenA) gene sequencing. A total of 11 species of filamentous fungi from three Ascomycota genera (Aspergillus, Penicillium, and Cladosporium) were recorded from deep-sea benthic communities that formed on the Piip submarine volcano. Levels of the major fatty acids in strains of these fungi were determined.The taxa found are typical of both deep-sea habitats and deep-sea volcanoes. However, the absence of some genera (Acremonium, Aureobasidium, Alternaria …) indicates the weakening of the influence of the volcano on the slopes and base.

First report of Nigrospora lacticolonia causing leaf spot of Bougainvillea spectabilis in China

Bougainvillea spectabilis is an ornamental shrubby vine widely distributed in tropical and subtropical regions of China. In September 2020, a leaf spot of B. spectabilis was observed on Yongxing Island of Sansha City, Hainan Province. At a disease incidence of >30%, this leaf spot seriously reduced the ornamental value of affected plants. The typical symptoms included lesions consisting of circular or subcircular, gray pale centers with dark brown borders surrounded by diffused yellow margins. Fungal cultures isolated from colonies growing from surface-sterilized leaf tissues on potato dextrose agar exhibited similar morphological characteristics. The fungus was identified as Nigrospora lacticolonia based on its morphological characteristics and phylogenetic analysis using the internal transcribed spacer (ITS) region of rDNA, β-tubulin (TUB2) and translation elongation factor 1-alpha (TEF1-α) gene sequences. Pathogenicity tests showed that the fungus could infect B. spectabilis, and it was successfully re-isolated from inoculated leaf tissues, fulfilling Koch’s postulates. This is the first report of N. lacticolonia causing leaf spot on B. spectabilis.