Neopestalotiopsis Clavispora Research Articles

Sustainable management of blister and grey blight diseases of tea using antagonistic and plant growth promoting microbes in Western Ghats of India

The blister and grey blights are the devastating leaf diseases in tea. The severity of blister and grey blight diseases in tea ranged from 22% to 68% and 11.41%–57.02%, respectively in a survey conducted in Southern India during 2022–2023. The morphological and molecular identifications of pathogens revealed that Exobasidium vexans and Neopestalotiopsis clavispora were responsible for causing blister and grey blights on tea in southern India. The conventional management strategy with foliar spray of synthetic fungicides result in fungicidal residue in tea causes environmental pollution and health hazards to human. To overcome this the current study aims to manage the blister and grey blight diseases of tea through the microbial biocontrol agents. The bacterial biocontrol agents viz., Bacillus subtilis (BBV57), Bacillus amyloliquefaciens (TNAU), Pseudomonas fluorescens (Pf1) and the fungal antagonist, Trichoderma asperellum (TV 1) were evaluated for their efficacy against the both blight diseases. All the bio agents have significantly reduced the basidiospore germination and the mycelial growth of E. vexans and N. clavispora, respectively. Among the bio agents, B. amyloliquefaciens registered 88.36% reduction in the germination of E. vexans basidiospore and 85.56% of mycelial growth reduction of N. clavispora in vitro (P < 0.05). Under field conditions the combined soil and foliar application of bacterial antagonists viz., B. subtilis, B. amyloliquefaciens and P. fluorescens each with 1 ˟ 1011 CFU/ml @ 5% concentration at 7 days interval significantly reduced the blister blight incidence with 53.53% & 51.53% reduction over control and 51.53% & 43.65% of reduction over initial during first and second year, respectively. The combined application also reduced the grey blight incidence with 53.53% & 52.00% reduction over control and 56.81% & 47.65% of reduction over initial during first and second year, respectively. This treatments with bioagents were almost comparable with synthetic treatments, such as copper oxy chloride (0.25%) and hexaconazole (0.25%) with 64.63% and 64.76% reduction of blister and grey blight reduction in first year; 59.74%, 51.11% reduction in second year over control (P < 0.05), respectively. Thus, the application of B. subtilis, B. amyloliquefaciens and P. fluorescens can be recommended for the sustainable management of tea blister and grey blight diseases in tea gardens.

The Preliminary Analysis of Flavonoids in the Petals of Rhododendron delavayi, Rhododendron agastum and Rhododendron irroratum Infected with Neopestalotiopsis clavispora.

The petal blight disease of alpine Rhododendron severely impacts the ornamental and economic values of Rhododendron. Plant secondary metabolites play a crucial role in resisting pathogenic fungi, yet research on metabolites in alpine Rhododendron petals that confer resistance to pathogenic fungi is limited. In the present study, the secondary metabolites in Rhododendron delavayi, R. agastum, and R. irroratum petals with anti-pathogenic activity were screened through disease index analysis, metabolomic detection, the mycelial growth rate, and metabolite spraying experiments. Disease index analysis revealed that R. delavayi petals exhibited the strongest disease resistance, while R. agastum showed the weakest, both under natural and experimental conditions. UHPLC-QTOF-MS/MS analysis identified 355 and 274 putative metabolites in positive and negative ion modes, respectively. The further antifungal analysis of differentially accumulated baicalein, diosmetin, and naringenin showed their half-inhibitory concentrations (IC50) against Neopestalotiopsis clavispora to be 5000 mg/L, 5000 mg/L, and 1000 mg/L, respectively. Spraying exogenous baicalein, diosmetin, and naringenin significantly alleviated petal blight disease caused by N. clavispora infection in alpine Rhododendron petals, with the inhibition rates exceeding 64%. This study suggests that the screened baicalein, diosmetin, and naringenin, particularly naringenin, can be recommended as inhibitory agents for preventing and controlling petal blight disease in alpine Rhododendron.

First Report of Neopestalotiopsis clavispora Causing Fruit Brown Spot of Pomegranate in China.

Pomegranate (Punica granatum) is an important fruit crop for therapeutic and food applications. In June 2022, brown spots were observed on the fruit surface of pomegranate cultivar named Guangyan in Mengzi (23°20'6''N,103°25'5''E), Yunnan, China. The early spots appeared as circular or irregular lesions, measuring 1~1.5 mm in diameter. They were light brown with a clear boundary between disease and healthy tissues. Over time, these spots developed into polygonal lesions covering the entire fruit surface. Eventually, the diseased fruits decayed, and more than 50% of fruits were infected in pomegranate orchards. The tissues from the interface between health and disease were cut down, immersed in 75% ethanol for 15 s, then 5% NaOCl disinfecting for 2 min, washed three times with sterile water, and the PDA cultured at 26 °C in an incubator under dark conditions. Twenty-five samples were collected for pathogen isolation, ten fungal isolates were obtained by single spore germination, and these isolates had similar morphological characters. The colonies were white with 81 mm diameter at 7 days of incubation, containing undulate edges with dense aerial mycelium. After 14 days, the black conidiomata formed superficially, gathering into black droplets. Conidiogenous cells were hyaline, short, and filiform. Conidia were fusiform, straight or slightly curved, and comprised five cells, 24.12 to 34.53 (x̄=29.78) μm × 4.21 to 12.15 (x̄=8.68) μm (n=50). The three median cells were 13.13 to 25.22 μm (x̄=18.54), dark brown, whose septa and periclinal walls were darker than the other two cells. The apical cells showed two to four appendages, 12.31 to 29.15 (x̄=21.56) μm. Only a single appendage was found on the basal cell, 2.34 to 7.16 μm. Based on morphological features, these isolates were identified as Neopestalotiopsis clavispora (Maharachchikumbura et al., 2012, 2014). Molecular identification of isolate YNSL-3 was performed by amplification and sequencing of ITS4/ITS5, BT2A/ BT2B and EF1-728F/EF-2, respectively (White et al. 1990, Glass et al.1995, Carbone et al. 1999, O'Donnell et al. 1998). These base sequences were deposited in GenBank with accession numbers OQ891378 (ITS), OR088917 (Tef) and OR513439(Tub), respectively. BLAST searches of the sequences revealed 100% (478/478 bp), 100% (484/484 bp), and 94.67% (426/450 bp) homology with those of N. clavispora NM16311a from GenBank (LC209216, LC209220, and LC209221), respectively. Phylogenetic analysis (IQ-TREE) by maximum likelihood method showed that the isolate YNSL-3 was clustered with N. clavispora. The pathogenicity was tested with the isolate of YNSL-3, YNSL-5 and YNSL-8 by detached assay. The fruit surface of pomegranate cultivar Guangyan was wounded with a sterilized needle. The mycelial blocks (5mm2) of isolates cultured on PDA for 7 days were attached to the points of inoculation. Controls were inoculated with sterile PDA agar. All inoculated fruits were maintained in a growth chamber at 26°C with 75% relative humidity. The test was performed thrice. The brown lesions were observed after 7 days, whereas the controls showed no symptoms. The same pathogens reisolated were identical to the original isolates based on morphological characterization and molecular analyses. N. clavispora could cause different diseases in many plants (Rajashekara et al. 2023, Loredana et al. 2020). To our knowledge, this is the first report of fruit brown spot on Punica granatum caused by N. clavispora in China. This finding will help improve management strategies against the fruit brown spots on P. granatum in China.

Isolation and biological activity of six new polyketide and terpenoid derivatives from Neopestalotiopsis Clavispora AL01

A fungus strain, Neopestalotiopsis clavispora AL01, was isolated from the leaf spot of the plant Phoenix dactylifera. Further chemical investigation of the fermentation extract of this strain afforded six new secondary metabolites (1–6), along with 11 known compounds (7–17) which included a new natural compound (7). Their structures were determined by extensive spectroscopic analysis including one-and two-dimensional (1D and 2D) NMR spectroscopy, high-resolution electrospray ionization mass spectrometry (HRESIMS), and ECD and NMR calculations. All compounds were evaluated for their phytotoxic activities. Among them, compounds 10, 12 and 13 exhibited phytotoxic activities against Nicotiana tabacum. Compound 3 exhibited weak antibacterial activity against methicillin-resistant Staphylococcus aureus, Micrococcus luteus and Vibrio harveyi. Taken collectively, these findings establish a solid research foundation for future investigations on bioactive natural products derived from phytopathogenic fungi.

Occurrence of Neopestalotiopsis clavispora Causing Leaf Blight on Acer oblongum in China.

Acer oblongum is native to Southwest China and is also distributed in Nepal and Northern India. It is an excellent garden ornamental tree species, suitable for solitary planting in courtyards and parks. From June to August 2022, severe leaf blight occurred on A. oblongum in Baihe Wetland Park (32°5'42" N, 112°28'13" E) in Nanyang City, China. The foliar disease rate reached 59% (n=100). Early signs were yellow spots on the leaves, mainly on the middle and edge parts. Then, the lesions gradually expanded, became amorphous, and turned yellowish brown, eventually led to necrosis on leaves and branches. Twenty diseased leaves were collected and the junction areas between infected and healthy tissues were cut into 5 x 5 mm2 pieces. The collected plant materials were sterilized in 75% ethanol and 1% NaClO for 30 s and 1 minute, respectively, followed by rinsing in sterile water, and placing on a potato dextrose agar (PDA) plate supplemented with 50 µg ml-1 streptomycin at 25 °C for 3 days. Colony edges were cut and transferred to new PDA plates for purification culture. A total of 18 purified fungal strains were obtained, which showed similar phenotypes in morphological characteristics. All colonies had spread radially with wavy surfaces, and dense cream to white aerial hyphae. After 14 days in culture, black fruiting bodies appeared. Conidia were fusiform to slightly clavate, with five cells and two or three setae, 4.2 to 7.9 μm × 17.5 to 25.4 μm in diameter (n = 100). The apical and basal cells and setae were colorless, three median cells were brown, and the middle cell was dark brown. Morphological characteristics of all 18 strains were consistent with the genus description of Neopestalotiopsis spp. (Maharachchikumbura et al. 2014). Further molecular identification showed that the ITS region sequences of all strains have extremely high homology with Neopestalotiopsis spp. The β-tubulin gene (TUB), and the translation elongation factor 1-alpha gene (TEF1) were amplified for molecular identification (Shu et al. 2020). The sequences of three representative strains (FE-05, 09, 16) from different regions were deposited in GenBank with accession Nos. OQ867279, OQ867288, OQ867289 (ITS), OQ870207, OQ870208, OQ870209 (TUB), and OQ870204, OQ870205, OQ870206 (TEF1). BLASTn analysis of these sequences showed 99 to 100% identity to Neopestalotiopsis clavispora strains (OK655673, MZ648263 for ITS, ON000362, MZ286974 fr TUB, MH423941, MK512481 for TEF1). These morphological features and molecular identification indicated that the pathogen has the same characteristics as N. clavispora. Pathogenicity was tested on ten healthy 3-month-old seedlings using the three representative strains through in vivo experiments. For each strain, the conidial suspension (106 conidia ml-1) in absorbent cotton balls (50 µl of inoculum) were inoculated onto the healthy leaves of two seedlings, while a total of two other plants were served with sterile water as a blank control. The plants were potted in a climate incubator at 28°C and a relative humidity of approximately 90%. Symptoms consistent with natural lesions were observed on the inoculated leaves after 5 days while the control plants remained healthy. The strains of N. clavispora were reisolated from the symptomatic inoculated leaves, fulfilling Koch's postulates. N. clavispora is known to cause disease in a variety of plants in China, such as Dendrobium officinale (Cao et al., 2022), Fragaria ananassa (Shi et al., 2022), and Garcinia mangostana (Qiu et al., 2019). To the best of our knowledge, this is the first report of N. clavispora causing leaf blight on A. oblongum in China. The yellowing and falling off of leaves would seriously affects the garden landscape. It is necessary to further clarify the host range of the pathogen to select appropriate landscape matching plants in future planning.

First report of leaf spot on Daphniphyllum macropodum caused by Neopestalotiopsis clavispora in China.

Daphniphyllum macropodum Miq., an evergreen arbor, is widely cultivated in southern China for its ornamental and medicinal value (Su et al. 2013). In October 2019, a severe leaf spot was observed on D. macropodum in Jinggangshan National Nature Reserve in Ji'an city, Jiangxi, China (114°06'23″E, 26°32'25″N). The plants were about 15 years old, and the disease incidence was estimated to be 15% (4/26 plants). The disease primarily appeared as small black spots on the leaves. At the late stage, the spots enlarged and coalesced into regular or irregular gray necrotic lesions with dark margins. We collected five samples per plant and total 20 samples were collected to isolated the pathogen strains. The margin of the diseased tissues was cut into 5 mm × 5 mm pieces; surface disinfected with 70% ethanol and 2% NaOCl for 30 s and 60 s, respectively; and rinsed thrice with sterile water. Tissues were placed on potato dextrose agar (PDA) and incubated at 25°C in the dark. Pure cultures were obtained by single-spore isolation method, and the representative isolates, JRM3, JRM6, and JRM8 were used for morphological studies and phylogenetic analyses. The colonies of three isolates grown on PDA were white, cottony, and flocculent, contained undulate edges with dense aerial mycelium on the surface at 25 °C. Conidiomata was black conidial masses on PDA. Conidia were 5-celled, clavate to fusiform, smooth, 19.3 to 24.4 long × 6.1 to 8.6 μm wide (n = 50). The 3 median cells were dark brown to olivaceous, the central cell was darker than the other 2 cells, and the basal and apical cells were hyaline. All conidia developed one basal appendage (3.4 to 8.3 μm long; n = 50), and 2 to 3 apical appendages (18 to 32 μm long; n = 50), filiform. The morphological characteristics of the isolates are comparable with those of the genus Neopestalotiopsis (Maharachchikumbura et al. 2014). The internal transcribed spacer (ITS) regions, β-tubulin 2 (TUB2) and translation elongation factor 1-alpha (TEF1-α) were amplified from genomic DNA for the three isolates using primers ITS1/ITS4, T1/Bt-2b, EF1-728F/EF-2 (Maharachchikumbura et al. 2014), respectively. The sequences of the isolates were submitted to GenBank (ITS, OQ372202 to OQ372204; TUB2, OQ390129 to OQ390131; TEF1-α, OQ390126 to OQ390128). A maximum likelihood and Bayesian posterior probability analyses using IQtree v. 1.6.8 and Mr. Bayes v. 3.2.6 with the concatenated sequences placed JRM3, JRM6, and JRM8 in the clade of N. clavispora. Based on the multi-locus phylogeny and morphology, three isolates were identified as N. clavispora. To confirm pathogenicity, eight healthy 10-year-old D. macropodum plants growing in the field were chosen, and 4 leaves per plant were wounded with a sterile needle and inoculated with 10 μL conidial suspension per leaf (106 conidia/ml). Eight plants inoculated with sterile water were used as control. All the inoculated leaves were covered with plastic bags to maintian a humidity environment for 2 days. The leaves inoculated with conidial suspension showed similar symptoms to those observed in the field, whereas control leaves were asymptomatic for 10 days. The same fungus were re-isolated from the lesions, whereas no fungus was isolated from control leaves. N. clavispora was determined as the pathogen of a variety of plant diseases, including Kadsura coccinea (Xie et al. 2018), Dendrobium officinale (Cao et al. 2022), Macadamia integrifolia (Santos et al. 2019). However, this is the first report of N. clavispora infecting D. macropodum in China. This work provided crucial information for epidemiologic studies and appropriate control strategies for this newly emerging disease.

Morphology, phylogeny and pathogenicity of fungal species associated with leaf blight and stem canker of Theobroma cacao in Malaysia

AbstractMalaysia stands prominently among Asia's key cocoa‐producing countries. In the cocoa season of 2022–2023, Malaysia demonstrated its contribution to the industry, with an estimated production of around 364,000 tonnes of cacao bean grindings. Nonetheless, fungal diseases pose undeniable challenges to the cocoa sector. Extensive sampling conducted between September 2018 and March 2019 across multiple states in Malaysia revealed concerning symptoms of leaf blight and stem canker affecting Theobroma cacao plants. The aim of this study was to identify and characterize fungal species associated with leaf blight and stem canker of T. cacao in Malaysia through morphological, molecular and pathogenicity analyses. Morphological and molecular phylogenetic analyses using multiple DNA regions (rDNA internal transcribed spacer [ITS], TEF1 and TUB2) were performed and identified 40 fungal isolates found in this study as Diaporthe tulliensis (17 isolates), Fusarium solani (seven isolates), Fusarium proliferatum (six isolates) and Neopestalotiopsis clavispora (10 isolates). Pathogenicity tests with mycelial plugs and wound treatments showed that D. tulliensis and N. clavispora were responsible for causing leaf blight whereas D. tulliensis, F. solani and F. proliferatum caused stem canker of T. cacao. The present study provides insights into disease aetiology and symptomatology that may be useful in planning effective disease management for the host plant.

Biocontrol Potential of Trichoderma asperellum CMT10 against Strawberry Root Rot Disease

Strawberry root rot caused by Neopestalotiopsis clavispora is one of the main diseases of strawberries and significantly impacts the yield and quality of strawberry fruit. Currently, the only accessible control methods are fungicide sprays, which could have an adverse effect on the consumers of the strawberries. Biological control is becoming an alternative method for the control of plant diseases to replace or decrease the application of traditional synthetic chemical fungicides. Trichoderma spp. are frequently used as biological agents to prevent root rot in strawberries. In order to provide highly effective biocontrol resources for controlling strawberry root rot caused by Neopestalotiopsis clavispora, the biocontrol mechanism, the control effects of T. asperellum CMT10 against strawberry root rot, and the growth-promoting effects on strawberry seedlings were investigated using plate culture, microscopy observation, and root drenching methods. The results showed that CMT10 had obvious competitive, antimycotic, and hyperparasitic effects on N. clavispora CMGF3. The CMT10 could quickly occupy nutritional space, and the inhibition rate of CMT10 against CMGF3 was 65.49% 7 d after co-culture. The inhibition rates of volatile metabolites and fermentation metabolites produced by CMT10 were 79.67% and 69.84% against CMGF3, respectively. The mycelium of CMT10 can act as a hyperparasite by contacting, winding, and penetrating the hyphae of CMGF3. Pot experiment showed that the biocontrol efficiency of CMT10 on strawberry root rot caused by Neopestalotiopsis clavispora was 63.09%. CMT10 promoted strawberry growth, plant height, root length, total fresh weight, root fresh weight, stem fresh weight, and root dry weight by 20.09%, 22.39%, 87.11%, 101.58%, 79.82%, and 72.33%, respectively. Overall, this study showed the ability of T. asperellum CMT10 to control strawberry root rot and its potential to be developed as a novel biocontrol agent to replace chemical fungicides for eco-friendly and sustainable agriculture.

Biological Control Activities of Rhizosphere Fungus Trichoderma virens T1-02 in Suppressing Flower Blight of Flamingo Flower (Anthurium andraeanum Lind.).

Flower blight caused by Neopestalotiopsis clavispora is an emerging disease of flamingo flower (Anthurium andraeanum Lind.) that negatively impacts flower production. The use of rhizosphere fungi as biocontrol agents is an alternative way to control this disease instead of using synthetic fungicides. This research aimed to screen the potential of rhizosphere fungi, Trichoderma spp., with diverse antifungal abilities to control N. clavispora and to reduce flower blight in flamingo flowers. A total of ten isolates were tested against N. clavispora by dual culture assay, and T1-02 was found to be the most effective isolate against N. clavispora, with inhibition of 78.21%. Morphology and molecular phylogeny of multiple DNA sequences of the genes, the internal transcribed spacer (ITS), translation elongation factor 1-α (tef1-α), and RNA polymerase 2 (rpb2) identified isolate T1-02 as Trichoderma virens. Sealed plate method revealed T. virens T1-02 produced volatile antifungal compounds (VOCs) against N. clavispora, with inhibition of 51.28%. Solid-phase microextraction (SPME) was applied to trap volatiles, and GC/MS profiling showed VOCs emitted from T. virens T1-02 contained a sesquiterpene antifungal compound-germacrene D. The pre-colonized plate method showed that T. virens T1-02 aggressively colonized in tested plates with inhibition of 100% against N. clavispora, and microscopy revealed direct parasitism onto fungal hyphae. Furthermore, the application of T. virens T1-02 spore suspension reduced the disease severity index (DSI) of flower blight in flamingo flowers. Based on the results from this study, T. virens T1-02 displays multiple antagonistic mechanisms and has the potential ability to control flower blight of flamingo flowers caused by N. clavispora.

The biological control effect of Bacillus cereus on strawberry leaf spot disease caused by Neopestalotiopsis clavispora

With their distinctive flavor, fragrance, and nutritional value, strawberry (Fragaria × ananassa Duch.) fruits are popular worldwide. However, strawberry is susceptible to pathogenic fungi in production, which can severely affect yields. In this study, we found a fungal disease in strawberries in Shenyang. The pathogen was identified from morphological characteristics and molecular data as Neopestalotiopsis clavispora. This fungus causes enormous economic losses in strawberry cultivation and production. It is essential to explore effective methods of controlling this disease, including chemical fungicides and biocontrol agents. This study evaluated the antifungal activity of 17 Bacillus strains isolated from soil against N. clavispora. These Bacillus strains showed significant antagonistic effects, with inhibition rates in the dual culture test of 50.22 %–79.48 %. Among these, Bacillus cereus (Bce-2) had the highest inhibition rate of 79.48 %. The ability of different working concentrations of Bacillus cereus culture solution to inhibit mycelium growth was variable, when the working concentration of Bacillus cereus culture solution was 0 %, 2 %, 4 %, 6 % and 8 %, the mycelium dry weight was 0.05–0.52 g. Furthermore, we optimized the fermentation method and evaluated the ability of the Bacillus cereus fermentation solution (BCFS) to control this disease in the field, the disease index of strawberry plants pre-treated with BCFS was reduced by 57.85 %. BCFS treatment improved the activities of peroxidase (POD), superoxide dismutase (SOD), catalase (CAT) and phenylalanine lyase (PAL). BCFS also prevents strawberry leaf spot disease caused by N. clavispora by increasing the expression of the following genes (WRKY22, WRKY29, ChiB, RbohD, HSP90, Rd19, PR1 and PP2C) in strawberry leaves. This study suggests that Bacillus cereus (Bce-2) may be effective in controlling N. clavispora.

First report of Neopestalotiopsis clavispora causing leaf spot on Phoebe bournei in China.

Phoebe bournei (Hemsl.) Yang is a typical evergreen broadleaf species widely distributed in subtropical China for its ornamental and economic value (Zhang et al. 2021). The wood of P. bournei is considered a good material for architectural decoration and furniture (Li et al. 2018). In June 2020, leaf spot symptoms were observed in Dexing (28°41'22.056″N, 115°51'52.524″E), Jiangxi province, China. Initial disease symptoms were small brown spots on the leaves. Then, the spots enlarged and coalesced into regular or irregular dark brown necrotic lesions with dark margins. Disease incidence in the field in Dexing was estimated 25%. Leaf pieces (5 × 5 mm) from the lesion borders were surface-sterilized in 70% ethanol for 30 s, followed by 2% NaOCl for 1 min, and then rinsed three times with sterile water. Tissues were placed on potato dextrose agar (PDA) and incubated at 25°C under a 14/10 h light/dark cycle for 4 days. Pure cultures were obtained by monosporic isolation, and the representative isolates, JX-N2, JX-N7, and JX-N11 were used for morphological studies and phylogenetic analyses. The colonies of three isolates grown on PDA were white, cottony, and flocculent, contained undulate edges with dense aerial mycelium on the surface. Conidia were 5-celled, clavate to fusiform, smooth, 18.7-24.6 × 5.9-8.8 μm (n = 100). The 3 median cells were dark brown to olivaceous, central cell was darker than other 2 cells, and the basal and apical cells were hyaline. All conidia developed one basal appendage (3.4-8.3 μm long; n = 100), and 2-3 apical appendages (17-30 μm long; n = 100), filiform. Morphological features were similar to Neopestalotiopsis sp. (Maharachchikumbura et al. 2014). The internal transcribed spacer (ITS) regions, β-tubulin 2 (TUB2) and translation elongation factor 1-alpha (TEF1-α) were amplified from genomic DNA for the three isolates using primers ITS1/ITS4, T1/Bt-2b, EF1-728F/EF-2 (Maharachchikumbura et al. 2014), respectively. All sequences were deposited into GenBank (ITS, OQ355048 - OQ355050; TUB2, OQ357665 - OQ357667; TEF1-α, OQ362987 - OQ362989). A maximum likelihood and Bayesian posterior probability-based phylogenetic analyses using IQtree v. 1.6.8 and Mr. Bayes v. 3.2.6 with the concatenated sequences (ITS, TUB2, TEF1-α) placed JX-N2, JX-N7, and JX-N11 in the clade of N. clavispora. Based on the multi-locus phylogeny and morphology, the representative isolates were identified as N. clavispora. The pathogenicity of three isolates were tested on six 9-year-old P. bournei plants, which were grown in the field. Three leaves per plant were wounded with a sterile needle (Φ=0.5 mm) and inoculated with 20 μL conidial suspension per leaf (106 conidia/mL). Another six control plants were inoculated with sterile water. Each leaf was covered with plastic bags to keep a humidity environment for 2 days. All the inoculated leaves showed similar symptoms to those observed in the field, whereas control leaves were asymptomatic for 9 days. N. clavispora was reisolated from the lesions, whereas no fungus was isolated from control leaves. N. clavispora can cause leaf diseases in a variety of hosts, including Machilus thunbergii (Wang et al. 2019), Fragaria × ananassa (Shi et al. 2022), Taxus media (Li et al. 2022). However, this is the first report of N. clavispora infecting P. bournei in China. This work provided crucial information for epidemiologic studies and appropriate control strategies for this newly emerging disease.

First report of Neopestalotiopsis clavispora causing foliar disease of rubber (Hevea brasiliensis) in the Philippines

AbstractIn September 2022, a leaf spot disease, which later coalesce and result into leaf defoliation of rubber was observed in Tungawan, Zamboanga Sibugay, Philippines. Morpho‐cultural and molecular characterization identified the pathogen as Neopestalotiopsis clavispora. Koch's postulates were fulfilled confirming its pathogenicity to rubber seedlings. To the best of our knowledge, this is the first report of N. clavispora infecting rubber in the Philippines.

First report of Neopestalotiopsis clavispora causing leaf blight on Aegicerascorniculatum in China.

Aegicerascorniculatum is an important mangrove plant that mainly grows in tropical and subtropical regions. Ithasimportantecological,economicandsocialbenefits (Bandaranayake 1998). In April 2021, a leaf disease on A.corniculatum was observed in Zhanjiang (21.21° N, 110.41° E), Guangdong province, China. Diseaseincidenceandseverity were 15%and 20% (n = 100 investigated plants), respectively. The disease symptoms on leaves primarily appeared as small brown spots, then enlarged and coalesced into regular or irregular gray necrotic lesions with dark margins. At the late stage of symptom development, black acervuli appeared on the necrotic lesions. Ten symptomatic leaves from 10 plants were collected. Small pieces of tissue (4 × 4 mm) were cut from lesion borders and were surfaced disinfected in 75% ethanol for 30 s, followed by 1 min in 1% NaClO, rinsed three times with sterile water, plated on potato dextrose agar (PDA), and incubated at 28°C. After 7 days, a total of 10 fungal isolates with 100% isolation frequency were obtained and three representative strains (THS-1, THS-2, and THS-3) were used for morphological and molecular characterization.Colonies were white with cottony aerial mycelium and irregular margins. Black viscous acervuli were scattered on the colony surface 10 days after incubation. Conidia were spindle shaped, five cells, four septa, 18.77-28.70 × 4.53-6.80μm (mean 23.13 × 5.14μm) in size (n = 50). Basal and apical cells were colorless while the three medium cells were dark brown and lightly versicolor. All conidia had one basal appendage of 3.51-7.27μm (mean 5.49 μm; n = 50) and two to three apical appendages of 15.80-33.64μm (mean 25.87 μm; n = 50). These morphological characteristics are consistent with those of Neopestalotiopsis clavispora(Maharachchikumbura et al. 2012). The ITS (OM698813-15), tub2 (OM810165-67) andtef1α(OM810186-88) sequences were 99.38%, 99.09%, and 99.17% identical to the type N.clavispora strain MFLUCC12-0281 (accession nos. JX398979, JX399014, and JX399045) through BLAST analysis, respectively. A phylogenetic tree was generated using the concatenated sequences of ITS, tub2, and tef1α. The result showed that these three isolates were clustered with N.clavispora strains including the type MFLUCC12-0281. To perform pathogenicity tests, 20 healthy potted seedlings of A.corniculatum (2-year-old) were selected. Ten surface-sterilized leavesof 10 seedlings were wounded and inoculated by spraying conidial suspension ( 105conidia/ml). The same number wounded leaves of the other 10 plants treated with sterile water served as controls. All plants were wrapped in polyethylene bags for 24 h and incubated at 28°C in a growth chamber (at 90% relative humidity). After 10 days, all the inoculated leaves showed similar symptoms to those observed in the field, whereas control leaves were asymptomatic. N. clavispora was reisolated from the lesions in terms of morphology and molecular characterization, whereas no fungus was isolated from the control leaves. The pathogenicity test was repeated three times under the same conditions. Thus, Koch's postulates were fulfilled. This pathogen has been reported on a wide host range worldwide, such as leaf spot on strawberry in China (Zhao et al. 2016) and twig blight on blueberry in Spain (Borrero et al. 2018). This is the first report of N. clavispora causing leaf blight on A.corniculatum in China.This study provides valuable information for the identification and control of the disease.

Inhibitory Activity and Mechanism of Action with Thymol against the Blueberry Pathogenic Fungi Caused by Neopestalotiopsis clavispora

Decay caused by Neopestalotiopsis clavispora is an important postharvest disease of blueberries that seriously affects the commercial value of blueberry fruit. In this paper, we studied the inhibitory activity and mode of action of thymol against the pathogenic fungus of blueberries caused by Neopestalotiopsis clavispora. The results demonstrated that thymol administration could limit mycelial growth in vitro; the inhibitory effect was positively connected with thymol mass concentrations, and the minimal inhibitory concentration (MIC) was 100 mg/L. Further investigations revealed that MIC thymol treatment dramatically reduced the germination of pathogenic spores and led to an increase in the conductivity of the pathogen, leakage of contents, and a decrease in pH. Propidium iodide (PI) staining experiments demonstrated that MIC thymol caused damage to mycelial cell membranes. Additionally, MIC thymol treatment promoted mycelium malondialdehyde content accumulation, inhibited superoxide dismutase (SOD) and catalase (CAT) enzyme activities, decreased adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) content and energy charge levels, and the fluorescence intensity of mycelium caused by MIC thymol treatment was significantly increased by the 2,7-Dichlorodi-hydrofluorescein diacetate (DCFH-DA) assay. The results of this study indicate that thymol suppresses the proliferation of Neopestalotiopsis clavispora by compromising the integrity of their cell membranes, promoting the accumulation of cellular reactive oxygen species (ROS), and interfering with energy metabolism.

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 Neopestalotiopsis clavispora causing leaf spot on Liquidambar formosana in China.

Liquidambar formosana Hance, a deciduous tree, is widely cultivated in China for its ornamental and afforestation value (Yin et al. 2021). In July 2019, leaf spot symptoms were observed with 20 to 30% disease incidence in Li shan forest farm (27°19'27.2″N, 115°32'51.08″E) in Ji'an city, Jiangxi province, China. Initial disease symptoms were small spots, which enlarged and circular to irregular, gray in the center, and dark brown to black circular on the lesion margin. Leaf pieces (5 × 5 mm) from the lesion borders were surfaced and sterilized in 70% ethanol for 30 s, followed by 2% NaOCl for 1 min, and then rinsed three times with sterile water (Si et al. 2022). Tissues were placed on potato dextrose agar (PDA) and incubated at 25°C. Pure cultures were obtained by monosporic isolation, and the representative isolates, FX-2, FX-5, and FX-9 were used for morphological studies and phylogenetic analyses. The colonies of three isolates on PDA grew fast, covering the entire plate with white cottony mycelia with black acervuli after 8 to 10 days. Conidia were 5-celled, clavate to fusiform, smooth, 19.6-24.2 × 6.2-8.5 μm (n = 100). The 3 median cells were dark brown to olivaceous, central cell was darker than other 2 cells, and the basal and apical cells were hyaline. All conidia developed one basal appendage (3.5-8.2 μm long; n = 100), and 2-3 apical appendages (18-31 μm long; n = 100), filiform. Morphological features were similar to Neopestalotiopsis sp. (Maharachchikumbura et al. 2014). The internal transcribed spacer (ITS) regions, β-tubulin 2 (TUB2) and translation elongation factor 1-alpha (TEF1-α) were amplified from genomic DNA for the three isolates using primers ITS1/ITS4, T1/Bt-2b, EF1-728F/EF-2 (Maharachchikumbura et al. 2014), respectively. All sequences were deposited into GenBank (ITS, ON622512- ON622514; TUB2, ON676532 - ON676534; TEF1-α, ON676529 - ON676531). A maximum likelihood and Bayesian posterior probability analyses using IQtree v. 1.6.8 and Mr. Bayes v. 3.2.6 with the concatenated sequences placed FX-2, FX-5, and FX-9 in the clade of N. clavispora. Based on the multi-locus phylogeny and morphology, three isolates were identified as N. clavispora. To confirm pathogenicity, 10 healthy 2-year-old seedlings, and 5 leaves per seedling were wounded with a sterile needle (Φ=0.5 mm) and inoculated with 200 μL conidial suspension per leaf(106 conidia/mL). Ten control plants were inoculated with ddH2O. All the inoculated leaves were covered with plastic bags and kept in a greenhouse at 26 ± 2 °C and RH 70%. All the inoculated leaves showed similar symptoms to those observed in the field, whereas control leaves were asymptomatic for 8 days. N. clavispora was reisolated from the lesions, whereas no fungus was isolated from control leaves. N. clavispora can cuase leaf diseases in a variety of hosts, including × Taxodiomeria peizhongii (Zhang et al. 2022), Macadamia integrifolia (Qiu et al. 2020), Dendrobium officinale (Cao et al. 2022). N. cocoes, N. chrysea, Pestalotiopsis neglecta and P. neolitseae were also reported to infect L. formosana (Fan et al. 2021). However, this is the first report of N. clavispora infecting L. formosana in China. This work provided crucial information for epidemiologic studies and appropriate control strategies for this newly emerging disease.

First report of Neopestalotiopsis clavispora Causing Leaf Spots on Garcinia mangostana in China.

Garcinia mangostana L. is a famous tropical fruit in Asia. In April 2021, a leaf disease on G. mangostana cv. Huazhu was observed in Zhanjiang (21.17° N, 110.18° E), Guangdong province, China. Symptoms was on new leaves of 2 year old plants. The spots were circular to irregular, gray in the center, and brown on the lesion margin. The disease incidence was estimated 25% (n = 500 investigated plants from about 50-ha). Twenty diseased leaves were collected from the orchard. The margin of the diseased tissues was cut into 2 mm × 2 mm pieces; surface disinfected with 75% ethanol and 2% sodium hypochlorite for 30 and 60 s, respectively; and rinsed thrice with sterile water. The tissues were plated onto potato dextrose agar (PDA) medium and incubated at 28 ℃. Twenty-eight isolates were obtained (isolation frequency = 28/4×20 = 35%). Single-spore isolation method was used to recover pure cultures for three isolates (GMN-1, GMN-2, and GMN-3) (Liu et al. 2021). The colonies were initially white with cottony aerial mycelium at 7 days on PDA. Then, they developed black acervular conidiomata at 10 days. Conidia were clavate to fusiform, four-septate, straight or slightly curved, and measured 16.5 to 21.4 µm long (average 19.5 µm; n = 40) × 4.5 to 6.5 µm wide (average 5.2 µm; n = 40). The three median cells were versicolored, whereas the basal and apical cells were hyaline. Conidia had a single basal appendage (4.5 to 5.5 µm long; n = 40) and three apical appendages (19.2 to 24.5 µm long; n = 40). The morphological characteristics of the isolates are comparable with those of the genus Neopestalotiopsis(Sajeewa et al. 2012). Molecular identification was performed using the colony polymerase chain reaction method with MightyAmp DNA Polymerase (Takara-Bio, Dalian, China) (Lu et al. 2012). Sequences were generated from the isolates using primers for the rDNA ITS (ITS1/ITS4), TEF1-α (EF1-728F/EF1-986R), and β-tubulin (T1/βt2b) loci (Sajeewa et al. 2012). The sequences of the isolates were submitted to GenBank (ITS, MZ026535-MZ026537; TEF, MZ032203-MZ032205; β-tubulin, MZ032206-MZ032208). The sequences of the isolates were 100% identical to the type strain MFLUCC12-0281 (accession nos. JX398979, JX399014, and JX399045) through BLAST analysis. The isolates clustered with N. clavispora (MFLUCC12-0280 and MFLUCC12-0281). The pathogenicity was tested in vivo. Individual plants (cv. Huazhu) were grown (n = 2, 1-1.5 year old) in a greenhouse at 24℃-30℃ with 80% relative humidity. Wounded leaflets were inoculated with 5-mm-diameter mycelial plugs or agar plugs (as control). Besides, sterile cotton balls were immersed in the spore suspension (1 × 105 per mL) and sterile distilled water (control) for about 15 s before they were fixed on the leaves for 3 days. One plant employed for each isolate with nine leaves. The test was performed thrice. Disease symptoms were found on the leaflets after 10 days, whereas the controls remained healthy. The pathogen was re-isolated from infected leaves and phenotypically identical to the original isolates to fulfill Koch's postulates. Neopestalotiopsis clavispora and Pestalotiopsis clavispora are synonyms. The fungus appeared to have a wide host range and distribution including in Thailand, Malaysia, North Queensland, and Australia (Sajeewa et al. 2012;Shahriar et al. 2022). Thus, this is the first report of N. clavispora causing leaf spot on G. mangostana in China. This finding will help improve management strategies against the leaf spots on G. mangostana in China.

Inhibitory Effect and Control Efficacy of Picoxystrobin against Neopestalotiopsis clavispora, Causing Vine Tea Leaf Blight

Vine tea (Ampelopsis grossedentata) is a traditional herb widely consumed in southwestern China that possesses paramount potential for human health. In 2021, the outbreak of typical leaf blight disease was observed in almost all vine tea plantations in Zhangjiajie of Hunan province, resulting in significant economic losses of vine tea production. In this study, we identified Neopestalotiopsis clavispora as the causal agent of vine tea leaf blight via its morphological characteristics and molecular identification. The sensitivity distribution of N. clavispora isolates to picoxystrobin were determined based on mycelial growth and spore germination inhibition assays. The EC50 values for mycelial growth ranged from 0.0062 to 0.0658 µg/mL, with a mean of 0.0282 ± 0.0148 µg/mL. The EC50 values for spore germination ranged from 0.0014 to 0.0099 µg/mL, and the mean value was 0.0048 ± 0.0022 µg/mL. Picoxystrobin increased fungal cell membrane permeability, but inhibited fungal ATP biosynthesis. Moreover, picoxystrobin exhibited good in planta control efficacy on vine tea leaves. Three picoxystrobin-resistant mutants were obtained in the current study, but no mutations were detected in the N. clavispora Cytb gene. Competitive ability assays showed that the conidium production and pathogenicity of all picoxystrobin-resistant mutants decreased as compared to their progenitors, indicating that picoxystrobin-resistant mutants suffer fitness penalty. These findings provide important evidence for picoxystrobin in vine tea leaf blight management and increase understanding of the resistance mechanism of picoxystrobin against N. clavispora.

Species of the Genera Neopestalotiopsis and Alternaria as Dominant Pathogen Species Attacking Mastic Trees (Pistacia lentiscus var. Chia)

Between 2018 and 2021, several mastic trees (Pistacia lentiscus var. Chia) sampled in the field and the nursery of the Chios Mastiha Growers Association (CMGA) were analyzed to determine the cause of dominant diseases. Symptoms included defoliation, leaf, and twig blight, wilting and/or apoplexy of trees and apoplexy of young hardwood cuttings. Moreover, brown discoloration had also been observed on older woody parts of the trees such as branches and tree trunks. Several pathogens have been isolated and identified as the causing agents. Neopestalotiopsis and Alternaria species were isolated consistently from necrotic tissues of mastic trees (branches, twigs, and leaves) in the field and the nursery. All fungal isolates’ pathogenicity was confirmed by applying Koch’s postulates on young mastic trees under glasshouse conditions. Fungal pathogens were identified by sequence analyses of the ITS, β-tubulin, and histone gene regions. Alternaria species were analyzed further by sequencing the endopolygalacturonase (endoPG) and the Alternaria major allergen (Alta1) genes. More specifically, the isolates were identified as Neopestalotiopsis clavispora, Alternaria arborescens, and A. alternata based on morphological features and sequence analyses. This is the first report of N. clavispora, A. arborescens, and A. alternata on P. lentiscus var. Chia.

First Report of Neopestalotiopsis clavispora Causing Postharvest Fruit Rot on Actinidia arguta in Liaoning Province, China.

Actinidia arguta, commonly called hardy kiwifruit or kiwiberry, is a perennial vine of Actinidiaceae Actinidia genus. Understanding the main pathogens that cause the fruit rot of A. arguta during storage is of great significance for finding strategies to prevent fruit rot. In September 2020, the A. arguta (Sieb.et Zucc.) Planch. ex Miq. LD133 was harvested from a farm in Dandong City, Liaoning Province, China (40°31'N, 124°20'E). After being stored at room temperature for about a week, the fruit rotted (no mechanical damage or wound). Initial symptoms were localized irregular spots, which then became soft and the spots connected into large, flaky, light brown lesions. Later symptoms were dark brown lesions and rot that affect the entire fruit. The strain that shows the same morphology as observed in the four decaying tissues was isolated on PDA. The strain was white, edge irregular and surface wavy, and the reverse side was pale yellow (Fig. S1A). The black viscous acervuli appeared on the surface of the mycelium after 7 to 10 days at 25℃ with 12 hours photoperiod. Conidia were fusiform to ellipsoid, straight to slightly curved, 5.7×25.9 µm (width × length), n=30, with five versicolor cells (three brown median cells, two hyaline cells on apical and basal). The apical cell generally contains one to four appendages on conidia (Fig. S1B). For identify the selected strains, three genetic regions (ITS, TUB and TEF 1-α) were used for amplification and sequencing. These sequences of pathogen shared 98 to 100% homology with Neopestalotiopsis clavispora. Then, a phylogenetic tree was constructed by the Bayesian algorithm using PhyloSuite (v1.2.2) (Zhang et al. 2020). Based on the morphological and molecular characterization, the pathogen was identified as Neopestalotiopsis clavispora (Chamorro et al. 2016). Next, pathogenicity of the screened strains was determined by wound inoculation method. Ripe healthy fruits are immersed in 1% NaClO, rinsed twice with sterile water. A sterile needle was used to penetrate 1-2mm of peel, and then inoculate hyphae (about 5mm in length, 1mm in diameter), and 10 µL of sterile water as a control. The treated fruits are stored in an artificial climate chamber (22°C, 70% relative humidity with 12 hours photoperiod). The inoculated fruit began to show signs of rot on the second day, and after 6 days, lesions similar to those found during storage of the pathogenic isolated fruit appeared. Similarly, 10 µL (106 conidia/mL) conidial suspension exhibited decay symptoms. Compared with conidial suspension as inoculum, hyphae has shorter incubation period and stronger pathogenicity to fruit (Fig. S2). The pathogen was re-isolated from these infected fruits and identified as N. clavispora, thus fulfilling Koch's postulates. N. clavispora has been reported causing root and crown rot on strawberry in Spain, Argentina, Uruguay and Italy (Chamorro et al. 2016; Gilardi et al. 2019; Machin et al. 2019; Obregon et al. 2018), and as a pathogen on blueberry in Spain and Korea (Borrero et al. 2018; Lee et al. 2019), and causing leaf spot on macadamia in Brazil (Santos et al. 2019), and causing leaf spot on Syzygium cumini in India (Banerjee and Rana 2020). However, to our knowledge, this is the first report of Neopestalotiopsis clavispora causing postharvest fruit rot of A. argute in the worldwide. The identification of the pathogen is of great significance for conducting research on A. argute fruit preservation to prolong its shelf life and improve its merchantability.