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.

First report of Neopestalotiopsis clavispora Causing guava scab in China.

Psidium guajava L. is widely cultivated in southern China. In May 2021, guava scab on cv. Zhenzhu was observed in Zhanjiang (21.18° N, 110.21° E), Guangdong province, China. Guava scab was corky with ovoid or round lesions on the surfaces of green fruits. Gradually the lesions sunk. Disease incidence was estimated as 85% in 500 investigated plants in about 50 ha. Twenty diseased fruits were collected from twenty trees in the field. From each fruit 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, successively; and rinsed thrice with sterile water. The tissues were plated onto potato dextrose agar (PDA) medium and incubated at 28 ℃. Thirty-four isolates were obtained. Single-spore isolation method (Liu et al. 2021) was used to recover pure cultures of three isolates (PGNC-1, PGNC-2, and PGNC-3) . The colonies were initially white with cottony aerial mycelium at 7 days on PDA. Then, these colonies form black acervular conidiomata at 10 days. Conidia were clavate to fusiform, four-septate, straight or slightly curved, and measured 15.8 to 21.2 µm × 4.5 to 6.5 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 were consistent with the description of Neopestalotiopsis clavispora (Maharachchikumbura et al. 2012). Molecular identification was performed using PCR 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 ß-tubulin (T1/ßt2b) loci (Maharachchikumbura et al. 2012). The sequences of the isolates were submitted to GenBank (ITS, OQ996557 to OQ996559; TEF, OR101037 to OR101039; ß-tubulin, OR100971 to OR100973). 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). N. clavispora and Pestalotiopsis clavispora are synonyms. The pathogenicity was tested in vivo. Plants (cv. Zhenzhu) were grown ( 3 years old) in a quarantine orchard at 25 â„ƒ to 32 â„ƒ with 60 to 80% relative humidity in May 2022. Disease-free green fruits were inoculated. 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 wounded fruits with transparent tape. Five fruits on one plant per isolate were inoculated. Five fruits on one plant severed as control. The test was performed thrice. Disease symptoms were found on the inoculated fruits after 20 days, whereas the controls remained healthy. The pathogen was re-isolated from infected fruits and was phenotypically identical to the original isolates thus fulfilling Koch's postulates. Neopestalotiopsis or Pestalotiopsis spp. were reported to be the causal agents of guava scab in Colombia and in Hawaii (Keith et al. 2006; Solarte et al. 2018). N. clavispora has been reported to cause disease in a broad range of hosts (Ge et al. 2009; Chen et al. 2018), but not in guava. This is the first report of N. clavispora causing guava scab in China. There would be no harvest if this disease is left unmanaged.

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, ß-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.

First Report of Neopestalotiopsis clavispora causing Leaf Spot on Coconut Seedling in Malaysia.

Coconut (Cocos nucifera) is a high economic value cash crop in Malaysia. In December 2021, irregular spots with dotted rust-like appearance were observed mainly on the tip of the leaves of MATAG variety coconut seedlings at the nursery in Perak state. More than 90% of the coconut seedlings surveyed were infected with leaf spot symptoms. These symptoms could bring huge economic losses due to the downgrade value of the seedlings. 15 symptomatic leaves were obtained from the nursery, 10 mm2 of cut leaves were disinfected with 10% sodium hypochlorite for 10 minutes and rinsed with sterile distilled water before plated on potato dextrose agar (PDA). A total of 4 single-spore isolates were obtained and were observed morphologically. The isolates had white cotton-like appearance with undulate edge. Black acervuli were seen after 7 days of incubation at 26 °C. The conidia were fusiform and contained five cells with four septate and three versicolor cells in between the apical and basal cell. The conidia were 17.2 µm long and 5.9 µm wide (n=30). Conidia consisted of two to three apical appendages and one basal appendage. These morphological characters were consistent with the original description of Neopestalotiopsis clavispora (Santos et al., 2019; Abbas et al., 2022). Species identification was done by amplifying internal transcribed spacer (ITS) region using primers ITS 4 and ITS 5 (White et al., 1990) and beta-tubulin (TUB2) using primers Bt2a and Bt2b (Glass & Donaldson et al., 1995) of the representative isolate LKR1, then sequenced. The 488 bp ITS and 409 bp TUB2 sequences were deposited in GenBank under the accession numbers ON844193 and OP004810, respectively. Isolate LKR1 shares 99.8% identity with the ITS sequence (MH860736.1) of the reference pathogenic N. clavispora strain CBS:447.73 and 100% identity with the TUB2 sequence (KM199443.1) of the reference pathogenic N. clavispora strain CBS 447.73. The phylogenetic analysis confirmed that the isolate LKR1 belonged to N. clavispora when a supported clade is formed with 98% and 94% bootstrap support for ITS and TUB2 respectively with other related N. clavispora. Pathogenicity test was conducted by using five replicates of 8 month old seedlings, they were incubated under greenhouse condition and were watered daily. The leaves of the seedlings were injured with sterile needles and were sprayed with conidial suspension (1 x 10^6 conidia/ml). The control plants were also injured but sprayed with sterile distilled water. After a month, signature symptoms of spots on the leaves appear but none on the control seedling. N. clavispora was successfully re-isolated only from the inoculated symptomatic leaves and identified morphologically. No fungus was re-isolated from the control seedlings. The result was consistent even after repeating the test one more time. N. clavispora has been reported causing leaf spot on Macadamia integrifolia (Santos et al., 2019), Phoenix dactylifera L. (Basavand et al., 2020) and Musa acuminata (Qi et al., 2022). N. clavispora has also been reported causing rust-like appearance of leaves on strawberry (Fragaria × ananassa Duch.) (Obregón et al., 2018). To our knowledge, this is the first report of N. clavispora causing leaf spot disease on coconut seedlings in Malaysia. Through the identification of N. clavispora as the causal agent of leaf spot on coconut, this can help coconut growers to tackle the disease problem earlier thus, preventing the disease from spreading until the adult phase.

First report of Neopestalotiopsis clavispora causing leaf spot on Photinia bodinieri in China.

Photinia bodinieri Lévl. is an evergreen broadleaf species widely cultivated in subtropical China as an ornamental value (Zhang et al. 2018). In July 2021, leaf spot symptoms were observed on the campus of Jiangxi Agricultural University (28°45'56″N, 115°50'21″E), Jiangxi province, China. The spots were circular to irregular, gray in the center, and dark brown on the lesion margin. The disease incidence was estimated 15%. 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 in the dark. Pure cultures were obtained by monosporic isolation, and the representative isolates, SN-3, SN-7, and SN-11 were used for morphological studies and phylogenetic analyses. The colonies of three isolates grown on PDA were white, cottony, and exhibited flocculent, contained undulate edges with dense aerial mycelium on the surface. Conidia were 5-celled, clavate to fusiform, smooth, 18.2-24.3 × 5.5-8.4 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. Conidia developed filiform appendages; one basal appendage (3.3-8.2 µm long; n = 100), and 2-3 apical appendages (16-29 µm long; n = 100). Morphological features were similar to Neopestalotiopsis sp. (Maharachchikumbura et al. 2014). Portions of internal transcribed spacer (ITS) regions, ß-tubulin 2 (TUB2) and translation elongation factor 1-alpha (TEF1-α) genes 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, OQ572345 - OQ572347; TUB2, OQ597847 - OQ597849; TEF1-α, OQ597844 - OQ597846). 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 SN-3, SN-7, and SN-11 in the clade of N. clavispora. Based on the multi-locus phylogeny and morphology, three isolates were identified as N. clavispora. Pathogenicity of the three isolates was verified on nine disease-free 7-year-old Photinia bodinieri plants, which were grown in the field. Two healthy leaves per plant were wounded with two pricks using a sterile needle (Φ=0.5 mm) and inoculated with 20 µL conidial suspension per leaf (106 conidia/mL). Another nine control plants were inoculated with sterile water. 36 leaves were used for the pathogenicity test of three isolates. All leaves were covered with plastic bags to maintain a humid environment for 2 days. The inoculated leaves showed similar symptoms to those observed in the field, whereas control leaves were asymptomatic after 10 days. The fungi were consistently reisolated only from the inoculated and symptomatic leaves, fulfilling Koch's postulates. N. clavispora can cause leaf diseases in a variety of hosts, including Kadsura coccinea (Xie et al. 2018), Photinia serratifolia (Yang et al. 2018), Camellia chrysantha (Zhao et al. 2020). Photinia spp. is an excellent landscape gardening plant, threatened with grey blight (Pestalotiopsis microspore) (Ye et al. 2022), anthracnose (Colletotrichum sp.) (Guan et al. 2013). However, this is the first report of N. clavispora infecting Photinia bodinieri 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 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, ß-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 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, ß-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 leaf spot on Elaeagnus pungens caused by Neopestalotiopsis clavispora in China.

Elaeagnus pungens Thunb. is a common traditional Chinese herbal medicine. It has high medicinal, edible, and ornamental value. In Sep. 2020, a leaf spot disease was found on E. pungens in the campus of Nanjing Forestry University, China (31°36'51"N, 119°11'8"E). The incidence rate was ca. 77%. The disease primarily appeared as small brown spots on the leaves. Then, the spots enlarged and coalesced into regular or irregular gray necrotic lesions with dark margins. At the late stage of symptom development, black spots (acervulus) appeared on the necrotic lesions (Fig. S2A-C). Eight symptomatic leaves were collected and surface-sterilized using 75% ethanol for 30 s followed by 1% NaClO for 1 min, and then washed three times in sterile distilled water. Cuttings (ca. 5×5 mm) were made from the margins of the lesions and placed on 2% of potato dextrose agar (PDA) in Petri plates and incubated at 25 ± 2 °C for 5 days. The isolation frequency of pathogens from diseased tissues was ca. 100%. A total of four fungal isolates 3-3-1, 3-3-2, 3-3-3, and 3-3-4 were obtained using the monosporic isolation method and stored in the Forest Pathology Laboratory at Nanjing Forestry University. For molecular identification, the internal transcribed spacer (ITS), partial translation elongation factor 1-alpha (TEF1-α), and partial ß-tubulin (TUB2) were amplified from the isolate 3-3-1, 3-3-2, 3-3-3 and 3-3-4, with the corresponding primer sets published in Maharachchikumbura et al. (2014). The amplicons of ITS (ON510047, ON510048, ON510070, and ON510069), TEF1-α (ON808445, ON808446, ON808447, and ON808448), and TUB2 (ON808449, ON808450, ON808451, and ON808452) generated from the isolate 3-3-1, 3-3-2, 3-3-3, and 3-3-4 were sequenced and deposited in GenBank. The ITS, TEF1-α, and TUB2 of the isolate 3-3-1 shared the same nucleotide sequences with the corresponding sequences of the isolate 3-3-2, 3-3-3, and 3-3-4. The ITS, TEF1-α, and TUB2 sequences showed 100%, 97%, and 99% similarity to Neopestalotiopsis clavispora MFLUCC12-0281 (ex-type), respectively. Phylogenetic analysis using concatenated sequences of ITS, TEF1-α, and TUB2 also showed that isolate 3-3-1, 3-3-2, 3-3-3, and 3-3-4 clustered monophyletically with N. clavispora, and supported with a high bootstrap value (80%) (Fig. S1). Since these four isolates were same species based on phylogenetic analysis, isolate 3-3-4 was randomly chosen for the pathogenicity test and morphological analysis. Colonies of the isolate 3-3-4 grown on PDA were white, cottony, and flocculent, contained undulate edges with dense aerial mycelium on the surface, and averaged 12.2 mm d-1 growth at 25 °C (Fig. S2F). Black conidiomata formed superficially, scattered over the PDA at two weeks post incubation, 170.15-1820.32 × 90.33-1230.12 µm (n = 109), and contained slimy black conidial mass (Fig. S2G). Conidiogenous cells were pear-shaped to cylindrical, transparent, and colorless to pale yellow with smooth cell walls (Fig. S2H). Conidia were spindle shaped, five cells, four septa, 18.46-25.9 × 5.3-9.37 µm, (av ± SD = 23.31 ± 1.81 × 7.33 ± 1.07 µm, n = 34) (Fig. S2I). Apical and basal cells were lighter in color, mostly hyaline, and the middle three cells were darker in color, mostly brown. The apical cell showed two to three colorless, transparent unbranched accessory filaments, 9.68-30.59 µm in length, (av ± SD = 20.57 ± 4.52 µm, n = 95), whereas the basal cell only a single appendage, 3.52-9.4 µm in length, (av ± SD = 5.32 ± 1.29 µm, n = 34) (Fig. S2I). These morphological characteristics were similar to N. clavispora described by Daengsuwan et al. (2021). Based on phylogenetic analysis and morphological characteristics, isolate 3-3-1, 3-3-2, 3-3-3, and 3-3-4 were identified as N. clavispora. Healthy potted seedlings of E. pungens (63-85 cm in height, 0.7-1.6 cm in diameter) were selected for the pathogenicity test in vivo. The surface-sterilized leaves were wounded with sterilized needles (1 mm in dia.) and inoculated with mycelial plugs and conidial suspensions, respectively. One part of the leaves were inoculated with mycelial plugs (5 mm in dia.) of isolate 3-3-4. The other part of the leaves were inoculated with 10 µL of conidial suspensions (1×106 spores mL-1). The inoculated plants were kept in a growth chamber at ca. 25 ± 2 °C and ca. 90% RH under a 12-h photoperiod. PDA discs without fungi and sterilized dH2O were used as controls, respectively. All experiments were repeated twice, and each treatment had six replicates at least. After 10 and 12 days post-inoculation, the necrotic lesions appeared on the leaves inoculated with the mycelial plugs and conidial suspensions of the isolate 3-3-4, respectively (Fig. S2D and E). However, no lesions were found on the plants inoculated with PDA discs and dH2O (Fig. S2D and E). Fungal isolates were re-isolated from the infected leaves and shared similar morphological characteristics of colonies and conidia with the original one. Thus, Koch's postulates were fulfilled. Neopestalotiopsis clavispora was determined as the pathogens of a variety of plant diseases such as leaf spot on Taxus chinensis, gray blight on Camellia sinensis, and root and crown rot on strawberry (Kirschbaum et al., 2018; Wang et al., 2019a, b). To our knowledge, this is the first report of leaf spot caused by N. clavispora on E. pungens worldwide. The discovery will be helpful for monitoring and control of this disease in the future.

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 ß-tubulin (T1/ßt2b) loci (Sajeewa et al. 2012). The sequences of the isolates were submitted to GenBank (ITS, MZ026535-MZ026537; TEF, MZ032203-MZ032205; ß-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 â„ƒ-30 â„ƒ 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.

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.

First report of Taxus media branch blight caused by Neopestalotiopsis clavispora in China.

Taxus media Rehder is a high-value tree species and can be used for landscape afforestation and medicinal purposes considering its beautiful shape and secondary metabolite paclitaxel. In May 2019, branch blight of T. media was observed in plantations in Hangzhou, Zhejiang province, China. The lesions were brown, necrotic and sunken, and the branches were withering. Plants from five plantations were surveyed, and the disease incidence was 60%. Necrotic tissues from 5 plans from each plantation were cut into small pieces (5×5 mm), surface disinfected with 75% alcohol for 30 s and 2% sodium hypochlorite solution for 2-3 min, washed twice with sterile water, then dried on sterile filter paper. Branch pieces were aseptically transferred to potato dextrose agar (PDA) plates and incubated at 25℃ in the dark for 3 days. Five purified fungal cultures were obtained and the representative isolate (LA-01) that grew slightly more vigorously under the same condition was selected for morphological characterization and DNA sequence comparison. The colony was initially white and cottony, with circular growth, undulated edge, wavy surface and pale beige on the back. The conidia were long spindle-shaped, straight or slightly curved, 21-27 × 6-8 µm (n=50) in overall shape. They contained 5 cells with those at the apex and base hyaline. The second and third cells from the apex were darker (brown) than the fourth cell from the apex (pale brown). Two or three (most frequently observed) hyaline appendages were apparent at the apex, 16-31 µm in length. Morphological characteristics of the obtained isolate were consistent with those in the genus of Neopestalotiopsis (Solart et al. 2018). DNA of LA-01 was extracted from fresh mycelia using Minibest Universal Genomic DNA Extraction Kit (Takara, Toyoto, Japan) according to the manufacturer's instructions. The partial regions from the internal transcribed spacer (ITS), ß-tubulin gene (TUB2), and translation elongation factor subunit 1-a gene (TEF1) were amplified and sequenced using universal primer pairs ITS5/ITS4, BT2A/BT2B, and EF1-526F/EF1-1567R (Maharachchikumbura et al. 2012), respectively. The resulting nucleotide sequences were individually subjected to BLAST searches in GenBank. The nucleotide sequences of ITS (MZ148633, 554 bp), TUB2 (MZ286974, 473 bp ), TEF1 (MZ286975, 1030 bp) of the isolate LA-01 showed 99.64% similarity to ITS sequence (MW512858.1), 99.35% similarity to TUB2 sequence (MH423961.1), 98.18% similarity to TEF1 sequence (KU096881.1). The multi-locus phylogenetic tree was constructed based on the combined ITS-TUB2-TEF1 sequences using MEGA 7 using the neighbor-joining method, indicating that LA-01 represents Neopestalotiopsis clavispora. Inoculation was done on branches of ten T. media plants in the field to verify the pathogenicity of LA-01. Two healthy branches of each plant were surface sterilized with 75% ethanol allowed to dry, and two small wounds on each branch were obtained by puncturing with a sterilized needle. One wound was inoculated with 5-mm-diameter PDA discs excised from 7-day-old cultures, and wrapped with parafilm; the other inoculated with sterile PDA plugs as control, and wrapped with parafilm. Disease symptoms developed on all N. clavispora-inoculated wounds 3 days after inoculation. The yellow brown lesions became apparent 20 days later, whereas the control wounds remained asymptomatic. The fungus N. clavispora was reisolated from the diseased lesions using the same methods above, fulfilling Koch's postulates. Previously, the species was reported to cause leaf spot disease on another Taxus tree species T. chinensis in China (Wang et al. 2019). This is the first report of T. media branch blight caused by N. clavispora worldwide. Special attention should be paid to the damage it may cause to T. media producers, and precautionary monitoring on T. media population will be a significant part of the conservation efforts in China.

First report of Neopestalotiopsis clavispora causing leaf spot disease on banana (Musa acuminate L.) in China.

Banana (Musa acuminate L.) is an important tropical fruit in China. In October 2020, a new leaf spot disease was observed on banana plants at an orchard of Zhenkang county (23°45'23.46″ N, 98°48'46.52″ E), Lincang city, Yunnan province, China. The disease incidence was about 1%. The leaf spots occurred sporadically and the percentage of the leaf area covered by lesions was less than 5%. Symptoms on the leaves were initially small, irregular, reddish-brown spots that gradually expanded to fusiform-shaped lesions with a yellow halo and eventually become necrotic, dry, and cracked. To isolate the pathogen, thirty symptomatic leaves (15 mm2) from five plants were surface disinfected in 70% ethanol (10 s) and 0.8% NaClO (2 min), rinsed in sterile water three times, and transferred to potato dextrose agar (PDA) at 28°C for 5 days. Twenty-five colonies formed on the PDA plates were white with cottony aerial mycelium, round with a light orange underside. Abundant black globular acervuli semi-immersed on PDA were observed after a week. Conidia were straight or slightly curved, clavate to spindle, five cells, four septa with dimensions of 17.49 to 34.51 µm × 4.24 to 7.28 µm (avg. 23.83 × 5.62 µm; n=50). The apical and basal cells were hyaline, whereas the three median cells were dark brown. Conidia had a single basal appendage with lengths of 2.95 to 17.7 µm (avg. 7.18 µm; n=50) and two to three apical appendages with lengths of 10.7 to 53.84 µm (avg. 17.36 µm; n=50). These morphological characteristics are consistent with those of Neopestalotiopsis spp. (Maharachchikumbura et al. 2014). To confirm species, single-spore cultures of two representative isolates CATAS-102001 and CATAS-102002 were selected for further identification. The internal transcribed spacer (ITS) region, translation elongation factor 1-α (TEF1-α) and ß-tubulin (TUB2) genes of the two isolates were amplified with primers ITS1/ITS4 (White et al. 1990), EF1-728/EF2 (O'Donnell et al. 1998; Carbone and Kohn, 1999) and T1/Bt2b (Glass and Donaldson, 1995; O'Donnell and Cigelnik, 1997), respectively, and sequenced. The sequences were deposited in GenBank (ITS: OM281005 and OM281006; TEF1-α: OM328820 and OM328821; TUB2: OM328818 and OM328819). A maximum likelihood phylogenetic tree was constructed using the MEGA 7.0 (Kumar et al. 2016) based on the concatenated sequences ITS region, EF1-α and TUB2 gene, and the cluster analysis placed the representative isolates CATAS-102001 and CATAS-102002 within a clade comprising Neopestalotiopsis clavispora. The pathogenicity of two isolates was conducted on six 7-leaf-old banana seedlings. Two leaves from each potted plants were stab inoculated by puncturing into 1-mm using a sterilized needle, and stabbing three points at both sides of leaf midrib, and then placing 10 µl conidial suspension (1×106 conidia/ml) on one side of wounded points and the other side of wounded points were inoculated with sterile water as control. Inoculated plants were kept inside a plastic bag for 72 h and maintained in the greenhouse (12 h/12 h light/dark, 28°C, 90% relative humidity). The experiments were repeated twice. Irregular necrotic lesions on inoculated leaves appeared 7 days after inoculation, whereas controls were asymptomatic. The fungus was recovered from inoculated leaves, and its taxonomy was confirmed morphologically and molecularly, fulfilling Koch's postulates. Neopestalotiopsis clavispora has been reported to cause leaf spot on Mangifera indica (Shu et al. 2020), Macadamia integrifolia (Santos et al. 2019) and Ligustrum lucidum (Chen et al. 2020). To our knowledge, this is the first report of N. clavispora on banana in China. The identification of N. clavispora as the causal agent of the observed leaf spot disease on banana is critical to the prevention and control of this disease in the future.

Red Foliage Blight of × Taxodiomeria peizhongii Caused by Neopestalotiopsis clavispora Newly Reported in China.

× Taxodiomeria peizhongii Z.J. Ye, J.J. Zhang & S.H. Pan is a hybrid of Taxodium mucronatum and Cryptomeria fortunei. It can adapt to various site conditions and has a good saline-alkali tolerance, which is a unique tree species in eastern China. In August 2020, a red foliage blight with an incidence of 70% (105/150 plants) was found on the leaves of × T. peizhongii in a nursery, Shanghai, China (121°21'12"E, 31°41'56"N). It developed from apical leaves of branches downwards. The infected leaves became reddish brown and withered. Fresh specimens were collected from 3 infected trees. Small samples (3-4 mm2) from lesion margins were sterilized, plated on potato dextrose agar (PDA) and incubated at 25°C. Nine isolates of the same fungus were obtained. Three representative isolates (DFS1-3, DFS1-8, and DFS1-9) were used for morphological and molecular studies and deposited in the China's Forestry Culture Collection Center (cfcc57401 to cfcc57403). The colonies of three isolates on PDA grew fast, covering the entire plate with white cottony mycelia in 7 days. Acervuli of DFS1-3 were 618-996 × 586-945 µm (n = 50). Conidiogenous cells were 4.4-9.8 µm (n = 50) long. Conidia were 5-celled, clavate to fusiform, smooth, 19-24 × 6.4-8.8 µm (n = 50). 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 µm long; n = 50), and 2-3 apical appendages (15-30 µm long; n = 50), filiform. The morphological characters of DFS1-8 and DFS1-9 were almost identical to DFS1-3. Based on morphological studies, the isolates were Neopestalotiopsis sp.. The DNA of 3 isolates was extracted. The internal transcribed spacer region (ITS), ß-tubulin 2 (TUB2) and translation elongation factor 1-alpha (TEF1-α) loci were amplified using the primer pairs ITS1/ITS4, T1/Bt-2b, EF1-728F/EF-2. BLAST result showed that ITS of the three isolates were identical to Pestalotiopsis sp. at a high level (greater than 99%), and TUB2, TEF1-α were highly similar with Neopestalotiopsis sp. (greater than 99%). The sequences were deposited in GenBank [Accession Nos. OM188301 and OM222696 to OM222697 for DFS1-3; OM188303 and OM222698 to OM222699 for DFS1-8; OM188302 and OM222700 to OM222701 for DFS1-9]. 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 (ITS, TUB2, TEF1-α) clustered 3 isolates together with N. clavispora including type isolate (MFLUCC 12-0281). Based on the morphology and phylogeny, the fungus was N. clavispora. To confirm pathogenicity, 9 healthy 2-yr-old seedlings, and 10 leaves per seedling were wounded with a sterile needle and inoculated with conidial suspension (106 conidia/mL). Three control plants were sprayed with sterile water. Seedlings were covered with plastic bags after inoculation and kept in a greenhouse at 25 ± 2°C and RH 80%. Seven days after inoculation, all inoculated leaves were reddish brown and withered like those observed in the field, whereas the control plants remained symptomless. N. clavispora was successfully reisolated from the infected tissues. This pathogen has been reported to cause leaf blight on many other hosts, such as Ligustrum lucidum and macadamia, but in recent years, the disease has also been reported on flowers, such as Anthurium. It has not been reported on Taxodium and Cryptomeria. This is the first report of N. clavispora infecting × T. peizhongii in the world. These data will help select appropriate fungicides for managing this newly emerging disease.

Three phytotoxins produced by Neopestalotiopsis clavispora, the causal agent of ring spot on Kadsura coccinea.

Phytotoxins are widely found in plant pathogens. In recent years, many diseases caused by Neopestalotiopsis clavispora have been reported. To better understand the pathogenicity of N. clavispora, a solid fermentation strategy was employed to isolate and identify virulence factors afritoxinone B, afritoxinone A and oxysporone. The phytotoxic activities of these toxins were evaluated. Oxysporone exhibited high levels of phytotoxic activity after 72 h and the lesion area ranged from 21.5-84.3 mm2 after 9 days of treatment. The phytotoxic activities of the other two compounds were lower than that for oxysporone. The phytotoxic activity towards non-host organisms was also assessed for the three analyzed compounds; phytotoxic activity was observed in each case. Based on these results, we conclude that oxysporone is the main virulence factor in N. clavispora. We also suggest that each of the three compounds were non-host-specific toxins (NHST). To our knowledge, this is the first study to analyze phytotoxins produced by N. clavispora.

Potentiality of Neopestalotiopsis clavispora ASU1 in biosorption of cadmium and zinc.

In this study, a fungal isolate was isolated from avocado fruit collected from a market in Makkah city, Saudi Arabia, and identified as Neopestalotiopsis clavispora ASU1. The biomass of Neopestalotiopsis clavispora ASU1 was used as a natural bio-sorbent for removal of Cd(II) and Zn(II) from aqueous solutions. Characterization of fungal biomass was performed using Fourier transform infrared spectroscopy, X-ray Diffractometer, and BET surface area. Different factors on Cd(II) and Zn(II) biosorption were studied to evaluate the maximum conditions for metals biosorption. The (qmax) for Cd(II) and Zn (II) by N. clavispora ASU1 calculated from the Langmuir adsorption isotherm was 185.3 ±â€¯0.25 and 153.8 ±â€¯0.21 mg/g, respectively. Based on r2, the equilibrium biosorption isotherms fitted well with Langmuir model than Freundlich isotherm. The adsorption kinetics was studied, and the biosorption followed to the pseudo-second-order model. Thus, the current study indicated that the biomass of N. clavispora ASU1 is an effective adsorbent for the removal of heavy metals from aqueous solutions.