First Report of Leaf Spot on Kidney Bean Caused by Nigrospora oryzae in China

Kidney bean (Phaseolus vulgaris L.) is a legume with high nutritional and economic value. This vegetable crop is widely cultivated in China, providing a year-round supply of young edible pods. In July 2020, a leaf spot disease on kidney bean cultivar ‘Dabailong’ was observed on a two-hectare field in Longli County (26°16′15.66″ N, 106°48′12″ E), Guizhou Province, China. Disease incidence was estimated to be nearly 50%. Foliar symptoms manifested as black circular spots, surrounded by a yellow halo and accompanied by white mycelium. To identify the pathogen, small portions of tissue (5×5 mm) from margins of leaf spots were cut from 20 symptomatic leaves, surface disinfected with 75% ethanol for 30 s, rinsed two times with sterile distilled water, dried on a sterile filter paper, and incubated on potato dextrose agar (PDA) at 28°C for 3 days. A total of 39 single-spore isolates were obtained. The colonies on PDA were fluffy, changing from white to gray or black with age, and reaching 7-cm diameter in 5 days at 28°C. Conidia were black, globose to subglobose, smooth, solitary, measuring 13.0 to 16.0 × 10.5 to 16.0 µm (n=30). Morphological characteristics were consistent with Nigrospora oryzae. In addition, the rDNA internal transcribed spacer (ITS), large subunit (LSU), β-tubulin (TUB) and translation elongation factor 1-alpha (TEF1) loci were amplified by PCR and sequenced (White et al. 1990, Glass and Donaldson 1995, O'Donnell et al. 1998; Carbone and Kohn 1999). The ITS, LSU, TUB and TEF1 sequences of two isolates, GUCC19-5105 and GUCC19-5192, were submitted to GenBank. BLASTn analysis of these sequences showed >98% homology with those of N. oryzae strain LC 7293 in GenBank (ITS, 99.80% (MZ145361 vs KX985931 – 498/499 bp) and 99.62% (MZ148445 vs KX985931 – 525/527 bp); LSU, 100% (MZ146317 vs KY806236 – 837/837 bp) and 99.76% (MZ148446 vs KY806236 – 847/849 bp); TUB, 98.72% (MZ329335 vs KY019601 – 386/391 bp) and 98.67% (MZ329337 vs KY019601 – 373/378 bp) and TEF1, 98.91% (MZ329336 vs KY019396 – 452/457 bp) and 98.89% (MZ329334 vs KY019396 – 444/449 bp) respectively). The phylogenetic tree of the combined 4 sequences showed that both isolates clustered with N. oryzae. Based on morphological characteristics and the multigene phylogenetic analysis, GUCC19-5105 and GUCC19-5192 isolates were identified as N. oryzae. Pathogenicity tests were performed twice by spraying conidial suspension (1×105 conidia/mL) of the two isolates (GUCC19-5105 and GUCC19-5192) on leaves of ten (five per isolate) healthy 5-week-old kidney bean cultivar ‘Dabailong’ plants. Two plants sprayed with sterile water served as controls. After inoculation, all the plants were kept moist in plastic bags for 24 hours and incubated in a greenhouse at 25°C for 20 days. Leaf spots similar to those observed in the field were observed 20 days post inoculation, but no lesions were observed on control plants. N. oryzae was reisolated from the infected tissues of inoculated kidney bean plants and the identity of the reisolated pathogen was confirmed as N. oryzae through morphology and sequencing ITS, LSU, TUB and TEF1 loci. In recent years, N. oryzae has been reported to infect a variety of plants such as Aloe vera, Citrullus lanatus and Costus speciosus (Begum et al. 2018; Chen et al. 2019; Sun et al. 2020). To our knowledge, this is the first report of leaf spot disease on kidney bean caused by N. oryzae in the world and provides a basis for diagnosticians and researchers to identify the disease and develop disease management strategies.

First Report of Leaf Blight Caused by Nigrospora oryzae on Poplar in China

Populus alba L. × P. berolinensis Dipp. (a hybrid poplar, ‘PaPb poplar’) exhibits fast growth and beautiful tree shape with high drought and cold tolerance, and is widely planted in the cities of Northeast China because of its ornamental and ecological value (Wang et al. 2008). In October 2020, an unknown leaf blight symptom was observed on the seedlings of ‘PaPb poplar’ at Shenyang Agricultural University (41°49′N, 123°34′E) located in Shenyang City, Liaoning Province, China. The disease incidence was 50% in a survey of 200 seedlings on the campus. The typical symptoms were brown-to-black, irregular-shaped lesions (Fig. 1A). To investigate the disease, five symptomatic leaves were collected, and pieces were cut at the margin of diseased and healthy tissue. These pieces were surface sterilized with 2% sodium hypochlorite for 2 min, rinsed three times with sterile distilled water, air dried, placed on potato dextrose agar (PDA) and incubated at 28°C. After 5 days of incubation, three isolates with similar morphological characteristics were observed. Isolate N03 was chosen and used for pathogen identification. The fungal colonies were initially white in color, and later turned gray to black (Fig. 1D). Conidia were single-celled, black, spherical or oblate in shape measuring 9.19 to 12.78 μm × 12.61 to 14.81 μm in diameter (n=40) (Fig. 1E). These were borne on hyaline vesicles at the tip of a conidiophore. Morphologically, the isolate N03 was identified as Nigrospora oryzae (Berk. and Broome) Petch (Wang et al. 2017). The genomic DNA was extracted with a SP Fungal DNA Kit (D5542-01, OMEGA). The internal transcribed spacer (ITS) region, translation elongation factor 1-alpha (TEF1-α), and partial beta-tubulin (TUB) genes were amplified using the primers ITS5/ITS4 (White et al. 1990), EF1-728F/EF-2R (Carbone and Kohn, 1999; O’Donnell et al. 1998), and Bt-2a/Bt-2b (Glass and Donaldson, 1995) primer sets, respectively. The PCR products of ITS, TEF1-α, and TUB were amplified, sequenced, and deposited in GenBank with the following accession numbers MZ148528, MZ182080, and MZ182079, respectively. BLASTn analysis of the ITS, TEF1-α, and TUB sequences had 99.3%, 99.8%, and 99.27% nucleotide identities to MK131325, KY019328, and KY019559, respectively. A phylogenetic tree based on combined ITS, TEF1-α, and TUB sequences was constructed using a Maximum Likelihood method with 1000 bootstraps showing that N03 was grouped with other N. oryzae isolates (Fig. 2). The fungus was identified as N. oryzae based on morphological characteristics and molecular analyses. Koch’s postulates were completed to confirm the pathogenity of N. oryzae on ‘PaPb poplar’. The N03 spore suspension (105 spores/mL) was used to inoculate detached leaves and field leaves in two experiments. The two experiments were repeated three times, respectively. In the detached leaf test, 10 healthy leaves collected from 1-year-old ‘PaPb poplar’ seedlings were inoculated with N03 by spraying with the spore suspension followed by incubation at 28°C on wet filter papers in a petri dish for 7 days. 10 leaves were sprayed with sterile water to save as the controls. For field leaf test, leaves of 5 plants were spray-inoculated with the spore suspension at the 4-week-old growth stage, and an additional 5 plants were sprayed with sterile water. Seven days after inoculation, brown-to-black, irregular-shaped lesions on the margin of leaves were observed on inoculated leaves but not on the controls (Fig. 1B and C). All detached leaves inoculated with N03 were symptomatic. In the field tests, symptom appeared on 20 of the 30 inoculated leaves. N. oryzae was re-isolated from all the inoculated detached leaves and inoculated plants, but not from the controls. N. oryzae is a known pathogen of several hosts, such as Costus speciosus (Koen.) Sm. and Mentha spicata L., but has not been reported on any species of Populus. To our knowledge, this is the first report of leaf blight of ‘PaPb poplar’ caused by N. oryzae in China and the world. This disease could affect growth and development of ‘PaPb poplar’ seedlings, and may cause economic losses in the future. Appropriate strategies should be developed to manage this disease.

Nigrospora oryzae Causing Leaf Spot on Asiatic Dayflower in Chongqing, China

Asiatic dayflower (Commelina communis L.) is an annual herbaceous weed that is distributed throughout China. A foliar disease on Asiatic dayflowers was discovered in one farm field in Dianjiang County, Chongqing, China (N30°3´22″, E107°18´5″) in summer, 2019. The disease incidence was observed on about 10% (13/127) of the plants. Symptoms appeared as round-shaped tan lesions (2-5 mm) in diameter that occurred randomly and irregularly on the whole leaves. The centers of lesions become grayish white with reddish borders as the disease progressed. The leaves with typical symptoms were detached and wiped with 70% ethanol for surface disinfestation before isolating the causal agent. Subsequently, three pieces (3-4 mm2) of tissue were taken from the margin of the leaf lesion, disinfested in 1.5% NaClO for 1 min, rinsed 3 times in sterilized distilled water, and placed onto Potato Dextrose Agar (PDA) medium containing 50 μg/ml each of kanamycin and ampicillin. A fungus was exclusively and consistently isolated from the disinfested leaf lesion sections. The colonies on PDA grew rapidly and covered the entire petri dish within 5 days at 28℃. Colonies were at first grayish white, cotton wool-like, round, with abundant aerial mycelium, and later turned black as conidia produced. The abundant conidia formed on PDA were initially yellow brown and gradually became black, oblate to ellipsoidal, smooth, single-celled, and ranged in size from 4 to 10 × 3.5 to 9 μm. They were borne on a colorless, hyaline, and inverted flask-shaped cell at the tip of each conidiophore. The morphology characteristics were consistent with those of Nigrospora spp. (Wang et al. 2017). Genomic DNA was extracted from one representative isolate NDJ0819. The amplification and sequencing of the gene fragments including the internal transcribed space (ITS) region of ribosomal DNA and beta-tubulin were performed using the primers ITS1/ITS4 (White et al. 1990) and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. Fragments of 536 bp for ITS and 408 bp for beta-tubulin were obtained. A phylogram of the combined ITS and beta-tubulin sequences reconstructed using the maximum likelihood bootstrapping method implemented in the software MEGA version 7.0 (Kumar et al. 2016) indicated that isolate NDJ0819 clustered with Nigrospora oryzae. Both ITS and beta-tubulin sequences were deposited into GenBank (accession no. MT140353 and MT157509, respectively). Pathogenicity test was performed by rub-inoculating needle-wounded leaves of three 4-week-old Asiatic dayflowers with spore suspension (2.6 × 106 conidia/ml) of NDJ0819 prepared in water containing 0.05% Tween-20, and holding plants at 28℃ in the growth chamber. The pathogenicity test was repeated twice. Brown, round-shaped lesions developed on leaves inoculated with spores at 15 days post-inoculation. However, the centers of the lesion did not become grayish white, compared to those of lesions seen in naturally infected leaves. No symptoms developed on leaves inoculated with sterilized distilled water. N. oryzae was re-isolated from the lesions. All results described above indicated that N. oryzae was responsible for the leaf spot of Asiatic dayflower. To our knowledge, this is the first report of N. oryzae causing leaf spot on Asiatic dayflower in China. Research into the potential use of N. oryzae as a candidate biological agent against the weed is worth being initiated.

PHYTOCHEMICAL STUDIES OF ENDOLICHENIC FUNGI ISOLATED FROM HYPOTRACHYNA INFIRMA (KUROK.) HALE

Objective: The aim of this study is to investigate of phytopharmaceutical importance of endolichenic fungi isolated from Hypotrachyna infirma (Kurok.) Hale. Methods: The lichen species were collected from Sholaiyar hills, Coimbatore and identified as Hypotrachyna infirma (Kurok.)Hale. From this lichen, 29 endolichenic fungi were isolated and 13 endolichenic fungi were identified. From the identified endolichenic fungi, 26 extracts were prepared by successive solvent extraction methods using Ethyl acetate and chloroform. Results: The phytochemical study revealed the presence of important constituents like Alkaloids, Tannins, Carbohydrates, Phenols, Protein, Terpenoids, Steroids, Glycosides Flavonoids and Saponins. From the 13 endolichenic fungi, only 5 endolichenic fungi (Nigrospora oryzae (Berkand Broome)Petch, Geotrichum candidum Link, Scytalidium lignicola pesante, Aspergillus oryzae(Ahlb.) cohn, Aspergillus niger Gr.) have more constitutents. These 5 endolichenic fungi have good results in Quantitative analysis also. Conclusion: Compared to ethyl acetate extracts Chloroform extracts showed very less concentration of the phytochemicals. From this study we conclutated Nigrospora oryzae (Berk and Broome) Petch gave the best results in both qualitative and quantitative compared to other endolichenic fungi.

First Report of Nigrospora oryzae Causing Leaf Spot on Ginger in China

Ginger (Zingiber officinale Rosc.) is an herbal crop widely grown in China for its medicinal and savory qualities of rhizomes. In August 2018, leaf spot symptoms were observed on ginger plants grown in a field in Nanning, Guangxi Province (E108°3'54", N23°14'48"). Disease incidence was above 50%, and in a Nanning field, rhizome yield loss was almost 30%. Early symptoms appeared as circular, necrotic areas that later developed into circular or irregular spots. The centers of the lesions were white and often surrounded by chlorotic halos (Figure S1A). In severe infections, the spots frequently coalesced, causing the entire leaf to become withered and curved. Small pieces (3 to 4 mm2) from the margin of infected lesions were surface sterilized in 75% ethanol for 40 s followed by 1% NaOCl for 90 s, placed on potato dextrose agar (PDA) and incubated at 28°C in the dark for 4 days. Hyphal tips from the leading edge of colonies were transferred to fresh PDA plates to obtain pure cultures. Fungal colonies were initially white, then turned black/grayish brown when maintained in the dark at 28°C after 5 days (Figure S1B). Conidia were single-celled, brown, or black, smooth, spherical, or subspherical with diameters varying from 9.5 to 15 μm (mean = 13.5 ± 0.72 µm, n = 50) (Figure S1C). Based on these morphological characteristics, the isolates were provisionally identified as Nigrospora oryzae (Ellis 1971; Hudson 1963). Genomic DNA was extracted from a representative isolate Sjb-2. The internal transcribed spacer (ITS) region, beta-tubulin (TUB2), and the translation elongation factor 1-alpha (TEF1-α) were amplified using primer pairs including ITS1/ITS4 (White et al. 1990), Bt-2a/Bt-2b (Glass and Donaldson 1995), and EF1-728F/EF1-986R (Carbone et al. 1999), respectively. The obtained ITS sequence (GenBank accession no. MW555242), TUB2 sequence (MZ048644), and TEF1-α sequence (MZ048645) showed >99% similarity with several GenBank sequences of N. oryzae (KF516962 for ITS; MK550707 for TUB2; and KY019425 for TEF1-α, respectively). Based on the combined sequences of ITS, TUB2 and TEF1-α sequences, a phylogenetic tree was constructed using the maximum likelihood method and confirmed that the isolates were N. oryzae (Figure S2). Pathogenicity of the isolate was confirmed by fulfilling Koch’s postulates. Agar blocks (3 mm diameter) containing a fungal mycelium were placed on detached healthy leaves of ginger. The leaves were then wrapped with sterile polyethylene and incubated in a greenhouse at 25°C with 60% RH. Within 7 days, symptoms appeared on inoculated leaves similar to spots observed in the field, whereas controls remained symptomless. The same pathogen was reisolated from the spots. Pathogenicity tests were performed twice with three replications, indicating that N. oryzae is responsible for leaf spot disease on ginger. The disease in ginger caused by N. oryzae had been reported in Southern Africa (Grech et al. 1989). To our knowledge, this is the first report of N. oryzae causing leaf spot of ginger in China. In the field, this pathogen can substantially affect ginger's health and rhizome yield if no effective control measures are implemented. Therefore, management of the disease should be further investigated to avoid major economic losses.

First report of Nigrospora oryzae causing wilt in summer cypress (Bassia scoparia) in Pakistan

Summer Cypress (Bassia scoparia) is a large annual herb belonging to the family Amaranthaceae native to Eurasia. It has been introduced in many other countries of the world. In Pakistan, summer cypress is also known as kochia and grown as an ornamental plant for its red fall foliage for landscapes. During October, 2017 a survey was conducted in Punjab Province, Pakistan, where 100 wilted plant samples were collected from 30 different plantations of Faisalabad district. Up to 50% loss of plantation was noted in all visited locations. Lower parts of the plants were affected first presenting with necrosis of leaf tips surrounded by a chlorotic zone (Fig. 1. A). Then necrosis of apical margins of the plant parts occurred, followed by stem discoloration and wilting of entire herbaceous branches, leading to the partial wilting of the plants. Ultimately, whole plant wilted and died, (Fig. 1. B) appearing as though they had been scorched. Diseased tissues from lower stem (crown portion) were sampled, surface sterilized in 70 % ethanol for 30 s, and cultured on to Potato Dextrose Agar (PDA) medium. Petri dishes were incubated at 25 ˚C with alternating 12-hour periods of light and dark. Frequently observed, fast growing whitish grey fungal cultures with black pin head points were obtained after 7 days (Fig. 1. C). Young conidia were one-celled, yellow to orange in color and turned brown to black (Fig. 1. D & E), ranged in size from 11 μm to 16 μm x 9.5 μm to 12 μm (Fig. 1. F), and were ellipsoidal at maturity (Fig. 2. A). Hyphae were branched, septate and dark brown in color while conidiophores were flexuous, branched and ranged between 3.5 μm to 4.5 μm in diameter and 14.5 μm to 26.5 μm in length. Based on morphology (Ellis, 1971), the pathogen was identified as Nigrospora oryzae and submitted to the Westerdijk Collection of Fungi, Netherland (CBS 146145/RNOEG30). Total DNA of isolate EG30 was extracted and portions of the Internal transcribed spacer (ITS) region and beta-tubulin (βt) gene were amplified using the universal primers ITS1F and ITS4 (White et al., 1990) and βt2a and βt2b (Glass and Donaldson, 1995). The generated ITS (GenBank Accession No. MG745331.1 491 bp) and βt (GenBank Accession No. MN629896 408 bp) sequences were searched against GenBank using BLASTn and were 99% homologous to ITS (KX986074 525 bp ; MN341493 550 bp) and 100% homologous to βt (MK262852 409 bp) gene region from Nigrospora oryzae (Wang et al., 2017; Zhang, 2019). For pathogenicity tests, ten healthy two-month-old summer cypress plants were inoculated by soil drenching of a spore suspension (106-107 spores/mL) of the fungal isolate EG30 while five plants were treated with sterilized water and used as control treatments. Plants were incubated at 60 to 75% relative humidity (RH) and 25 ˚C in a greenhouse. Leaf necrosis and partial to whole plant witling (Fig. 2. B & C) were observed in the inoculated plants after 21 days. No symptoms appeared in control plants. A fungus was re-isolated from the lower stem (crown portion) parts of the inoculated plants that was identical in morphology to isolate EG30. No fungus resembling EG30 was isolated from the control plants. To the best of our knowledge, this is the first report of summer cypress wilt caused by Nigrospora oryzae (Berk. and Broome) Petch, a known pathogen of several important crops in China, Australia, India, Canada, and Pakistan (Sharma et al., 2013).

Bioactive Secondary Metabolites from the Mangrove Endophytic Fungi Nigrospora oryzae

Mangrove forest has a distinctive habitat adapting with marine and terrestrial environment. Chemical investigation of the extract from mangrove endophytic fungi Nigrospora oryzae had resulted in the isolation of sterigmatocystin (1) and pestalopyrone (2). The structure of sterigmatocystin (1) and pestalopyrone (2) were elucidated using mass, UV and NMR spectrometers together with the comparison with the literature data. The study also showed that sterigmatocystin displayed moderate cytotoxicity but it could be further developed as antiviral and antibacterial agent based on the SAR information reported from its analogue and derivatives.

First Report of Leaf Spots caused by Nigrospora oryzae on Wild Rice in China

In recent years, wild rice (Oryza rufipogon Griff) has been widely cultivated because of its health-promoting effects. In May 2019, leaf spot lesions on cv. Haihong-12 were observed in Zhanjiang (20.93N, 109.79E), China. Leaf symptoms were yellow-to-brown, oval or circular with a very distinctive, large yellow halo. Black spores appeared on the leaves with advanced symptoms. The lesions coalesced, causing the entire leaf to become blighted and die. Disease incidence reached approximately 10% in the fields (8 ha) surveyed. Twenty leaves with symptoms were collected and cut into pieces of 2 ×2 cm in size. They were surface-disinfected with 75% ethanol for 30 s and 2% sodium hypochlorite (NaOCl) for 60 s, rinsed three times with sterile water, blotted dry on sterile paper, plated on potato dextrose agar (PDA) medium, and incubated at 28°C in the dark for 4 days. Ten pure cultures were obtained by transferring hyphal tips to new PDA plates, and monosporic cultures were obtained from three isolates (Nos-1, Nos-2, and Nos-3). Those isolates exhibited very similar morphological characteristics on PDA. Colony of isolate Nos-1 was white at the early stage and became dark gray after 7 days. Conidia were produced from clusters of conidiophores, single celled, black, smooth, spherical, and 9.5 to 14.2 µm (average 10.6 µm ± 0.42) in diameter. Morphological characteristics of the isolates matched the description of Nigrospora oryzae Petch (Wang et al. 2017). The ITS region was amplified using primers ITS1 and ITS4 (White et al. 1990). Nucleotide sequences of isolates Nos-1, Nos-2, and Nos-3 deposited in GenBank under acc. nos. MW042173, MW042174, and MW042175, respectively, were 100% identical to N. oryzae (acc. nos. KX985944, KX985962; and KX986007). A phylogenetic tree generated based on the ITS sequences and using a Maximum Likelihood method with 1,000 bootstraps showed that these three isolates from wild rice were grouped with other N. oryzae isolates downloaded from GenBank (bootstrap = 100%) but away from other Nigrospora spp. Pathogenicity test was performed with these three isolates in a greenhouse at 24 to 30°C. Approximately 50 seedling of wild rice cv. Haihong-12 were grown in each pot. At the 3-leaf stage, plants in three pots were inoculated with each isolate by spraying a spore suspension (105 spores/ml) until runoff. Three pots sprayed with sterile water served as the controls. Each 3-pot treatment was separately covered with a plastic bag. The test was conducted three times. Diseased symptoms were observed on the inoculated leaves after 10 days while no disease was observed in the control plants. Morphological characteristics and the ITS sequences of fungal isolates re-isolated from the diseased leaves were identical to those of N. oryzae. N. oryzae has been reported to cause leaf spot on O. sativa (Wang et al. 2017), but not on O. rufipogon. Thus, this is the first report of N. oryzae causing leaf spot of O. rufipogon in China. The finding provides the information important for further studies to develop management strategies for control of this disease.

FREQUENCY OF FUNGAL MYCOFLORA ON RICE

Introduction: Rice (Oryza sativa L.) is the second best essential cereal crop of the world as well as of Pakistan. There are various fungal pathogens that attack on rice and cause various rice diseases. Methods: Therefore, the present experiments were done to isolated different fungi associated with seeds, stems, and leaves of commercially grown rice varieties and of mainly isolated species. Results: Several fungi, namely, Magnaporthe oryzae, Curvularia lunata, Helminthosporium oryzae, Fusarium moniliforme, Alternaria alternata, Nigrospora oryzae, and Aspergillus niger were isolated from seeds, stems, and affected leaves of rice. Conclusion: In our research, the frequency of fungal mycoflora on rice has been calculated and the maximum isolates of Fusarium spp. have been found.