First report of Nigrospora sphaerica causing fruit dried-shrink disease in Akebia trifoliata from China

Akebia trifoliata, a recently domesticated horticultural crop, produces delicious fruits containing multiple nutritional metabolites and has been widely used as medicinal herb in China. In June 2020, symptoms of dried-shrink disease were first observed on fruits of A. trifoliata grown in Zhangjiajie, China (110.2°E, 29.4°N) with an incidence about 10%. The infected fruits were shrunken, colored in dark brown, and withered to death (Figure S1A, B). The symptomatic fruits tissues (6 × 6 mm) were excised from three individual plants, surface-disinfested in 1% NaOCl for 30s and 70% ethanol solution for 45s, washed, dried, and plated on potato dextrose agar (PDA) containing 50 mg/L streptomycin sulfate in the dark, and incubated at 25℃ for 3 days. Subsequently, hyphal tips were transferred to PDA to obtain pure cultures. After 7 days, five pure cultures were obtained, including two identical to previously reported Colletotrichum gloeosporioides causing leaf anthracnose in A. trifoliata (Pan et al. 2020) and three unknown isolates (ZJJ-C1-1, ZJJ-C1-2, and ZJJ-C1-3). The mycelia of ZJJ-C1-1, ZJJ-C1-2 and ZJJ-C1-3 were white, and formed colonies of approximate 70 mm (diameter) in size at 25℃ after 7 days on potato sucrose agar (PSA) plates (Figure S1C). After 25 days, conidia were formed, solitary, globose, black, shiny, smooth, and 16-21 μm in size (average diameter = 18.22 ± 1.00 μm, n = 20) (Figure S1D). These morphological characteristics were similar to those of N. sphaerica previously reported (Li et al. 2018). To identify species of ZJJ-C1-1, ZJJ-C1-2 and ZJJ-C1-3, the internal transcribed spacer (ITS) region, β-tubulin (TUB2), and the translation elongation factor 1-alpha (TEF1-α) were amplified using primer pairs including ITS1/ITS4 (Vilgalys and Hester 1990), Bt-2a/Bt-2b (Glass and Donaldson 1995), and EF1-728F/EF-2 (Zhou et al. 2015), respectively. Multiple sequence analyses showed no nucleotide difference was detected among genes tested except ITS that placed three isolates into two groups (Figure S2). BLAST analyses determined that ZJJ-C1-1, ZJJ-C1-2 and ZJJ-C1-3 had 99.73% to N. sphaerica strains LC2705 (KY019479), 100% to LC7294 (KY019397), and 99.79-100% to LC7294 (KX985932) or LC7294 (KX985932) based on sequences of TUB2 (MW252168, MW269660, MW269661), TEF-1α (MW252169, MW269662, MW269663), and ITS (MW250235, MW250236, MW192897), respectively. These indicated three isolates belong to the same species of N. sphaerica. Based on a combined dataset of ITS, TUB2 and TEF-1α sequences, a phylogenetic tree was constructed using Maximum likelihood method through IQ-TREE (Minh et al. 2020) and confirmed that three isolates were N. sphaerica (Figure S2). Further, pathogenicity tests were performed. Briefly, healthy unwounded fruits were surface-disinfected in 0.1% NaOCl for 30s, washed, dried and needling-wounded. Then, three fruits were inoculated with 10 μl of conidial suspension (1 × 106 conidia/ml) derived from three individual isolates, with another three fruits sprayed with 10 μl sterilized water as control. The treated fruits were incubated at 25℃ in 90% humidity. After 15 days, all the three fruits inoculated with conidia displayed typical dried-shrink symptoms as those observed in the farm field (Figure S1E). The decayed tissues with mycelium and spores could be observed on the skin or vertical split of the infected fruits after 15 days’ inoculation (Figure S1F-H). Comparably, in the three control fruits, there were no dried-shrink-related symptoms displayed. The experiment was repeated twice. The re-isolated pathogens were identical to N. sphaerica determined by sequencing the ITS, TUB2 and TEF-1α. Previous reports showed N. sphaerica could cause postharvest rot disease in kiwifruits (Li et al. 2018). To our knowledge, this is the first report of N. sphaerica causing fruits dried-shrink disease in A. trifoliata in China.

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First Report of Nectria haematococca Causing Stem Rot of Soybean in China

Plant Disease ◽

10.1094/pdis-07-11-0626 ◽

2012 ◽

Vol 96 (3) ◽

pp. 457-457 ◽

Cited By ~ 1

Author(s):

Y. Gai ◽

R. Pan ◽

D. Xu ◽

M. Deng ◽

W. Chen ◽

...

Keyword(s):

Elongation Factor ◽

Morphological Characteristics ◽

Its Region ◽

Stem Rot ◽

Universal Primers ◽

Conidial Suspension ◽

Fruiting Bodies ◽

Nectria Haematococca ◽

First Report ◽

Soybean Seedlings

In October 2010, soybean (Glycine max) plants growing in commercial soybean fields in Zengcheng City, Guangdong Province developed symptoms consisting of stem and root rot, yellowing, and defoliation of leaves. Reddish, spherical fruiting bodies appeared in lesions that developed on stems. Plants with symptoms were sampled from fields. Fruiting bodies were excised from diseased tissues. Microscopic examination revealed that they were perithecia, globose to pyriform, and measured 197 to 260 μm in diameter and 226 to 358 μm long. When squeezed gently, cylindrical to clavate asci, 7.2 to 9.6 μm in diameter and 75.4 to 92.0 μm long, containing eight ascospores were exuded from the perithecia. Ascospores were ellipsoid to obovate, two celled, slightly constricted at the septum, had longitudinal striations, and measured 4.9 to 6.0 μm in diameter and 10.6 to 15.0 μm long. The fungus was isolated from the basal stem tissues of diseased soybean plants and cultured on potato dextrose agar (PDA) medium amended with streptomycin sulfate. On PDA, the culture developed into blue-pigmented colonies with whitish mycelium that produced oval to cylindrical microconidia. Microconidia had 0 to 1 septum, ranged from 2.5 to 5.2 × 7.6 to 29.4 μm, and were produced on monophialides. Macroconidia were cylindrical to falcate, thick walled, 2 to 5 septa, and 3.5 to 6.0 × 25.4 to 66.8 μm. Chlamydospores were present and ranged from 6.8 to 13.6 × 5.5 to 9.5 μm. Orange-to-reddish perithecia were readily formed in old culture. These morphological characteristics were consistent with descriptions of Nectria haematococca (anamorph Fusarium solani) (1). The rDNA internal transcribed spacer (ITS) region and the fragment of translation elongation factor 1-alpha (EF1-α) genes of the fungus were amplified, respectively, with universal primers ITS1/ITS4 and ef1/ef2 primers and sequenced. BLAST searches showed that the ITS sequences of three isolates (GenBank Accession Nos. JN015069, JN190942, and JN190943) had 99% similarity with those of N. haematococca(GenBank Accession Nos. DQ535186, DQ535185, and DQ535183) and the EF1-α sequences of three isolates (GenBank Accession Nos. JN874641, JN874642, and JN874643) had 100% similarity with those of F. solani (GenBank Accession Nos. DQ247265 and DQ247327). Completion of Koch's postulates confirmed the pathogenicity of the isolates in a replicated experiment. Thirty-day-old soybean seedlings of cultivar Huaxia No. 3 were inoculated by soaking their root systems in a conidial suspension (106 conidia per ml) for 30 min and then transplanted in plastic pots (20 cm in diameter) and incubated at 25 ± 2°C in a greenhouse. Control plants were treated with sterile water in the same way. There were four plants per pot and there were six replicates for each treatment. Within 3 weeks, more than 70% of the inoculated plants exhibited symptoms of leaf yellowing, stem rot, and root rots while control plants were symptomless. N. haematococca was reisolated from the diseased plants. To our knowledge, this is the first report of N. haematococca causing stem rot of soybean in China and the first description of sexual reproduction of F. solani causing soybean stem rot in nature. This pathogen may pose a serious threat to soybean production in China where soybean is a main crop. Reference: (1) C. Booth. The Genus Fusarium. CAB International, Wallingford, UK, 1971.

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First report of blight on Fritillaria thunbergii caused by Fusarium oxysporum in Zhejiang Province, China

Plant Disease ◽

10.1094/pdis-11-21-2407-pdn ◽

2021 ◽

Author(s):

Yiwen Xu ◽

Zhenyan Cao ◽

Yihua Yang ◽

Xuping Shentu ◽

Xiaoping Yu

Keyword(s):

Early Stage ◽

Science And Technology ◽

Elongation Factor ◽

Morphological Characteristics ◽

Its Region ◽

Zhejiang Province ◽

Gastric Ulcers ◽

Conidial Suspension ◽

Sterile Water ◽

First Report

Fritillaria thunbergii Miq. (Zhe beimu), is an oldest known homeopathic traditional folk medicinal plant in Zhejiang Province, China. The bulbs are medicinally important curing cough, inflammation, gastric ulcers, hypertension, diarrhea, and bronchitis (Nile et al. 2021). In April of 2018 and 2019, symptoms of blight on F. thunbergii were observed with an incidence of 20–25% in Cixi city, Zhejiang Province. At the early stage of this disease, the stalk turned brown, then the whole bulbs rotted within a few days. Symptomatic bulbs were cut into small pieces (1.0 cm × 1.0 cm) and disinfected successively by submersion in 75% ethanol for 30 seconds and 1% NaClO for 30 seconds under aseptic conditions. After rinsing with sterile water three times and air drying, segments were placed on potato dextrose agar (PDA). After incubation at 28 ℃ for 7 days in the dark, the hyphae were observed white fluffy, spreading from the middle to the whole plate. Macroconidia were falciform with zero to four septa and (11.0-39.0) × (3.0-5.0) μm in size. Microconidia were fusiform with zero to two septa (4.0-7.0) × (2.6-3.0) μm in size. Based on morphological characteristics of macroconidia, and microconidia, isolates were identified as F. oxysporum (Lombard, L. et al., 2019). The internal transcribed spacer (ITS) region, translation elongation factor (EF-1α), and RNA polymerase second largest subunit (RPB2) gene were amplified and sequenced respectively using ITS1/ITS4, EF1/EF2 and 5f2/7cr primers (O’Donnell et al., 2010). BLASTN analysis of FUSARIUM-ID using ITS (Accession NO.MZ268594), EF-1α (Accession NO.MZ292517) and RPB2(Accession NO.MZ292516) showed 95.2%, 100%, and 99.11% identity to F. oxysporum species complex isolates NRRL43730, NRRL38599 and NRRL38302, respectively. Based on the morphological and molecular characters, the pathogen was identified as F. oxysporum. To verify pathogenicity, ten healthy F. thunbergii plants were used for inoculation tests. One milliliter of a conidial suspension (106 conidia ml-1) was pipetted onto the soil around the base of F. thunbergii plants per pot. Ten plants, which were treated with sterile water, were used as the control. All plants were maintained in a climatic chamber (26 ± 1 ℃, 70–80% relative humidity and a photoperiod of 16:8 [L: D] h). Seven days later, all inoculated plants showed typical symptoms of blight identical to those observed in the fields. Control plants remained symptomless and healthy. The pathogenicity analysis was repeated three times. Pathogens re-isolated from symptomatic plants were identified as F. oxysporum by morphology observation and sequence analysis. To our knowledge, this is the first report of blight caused by F. oxysporum on F. thunbergii in Zhejiang Province, China. Acknowledgments: The author(s) declare no conflict of interest. Funding: This work was supported by Zhejiang Provincial Program for Science and Technology Development (2017C32006, 2018C02030) and the Student Science and Technology Innovation Project of China Jiliang University (2021YW95). References: Nile et al. 2021.J. Food and chemical toxicology, 153:112289. Hami, A. et al., 2021. J. Scientific Reports. 3610.11.1. Lombard, L. et al., 2019. PERSOONIA, 43:1-47. O’Donnell, K., et al. 2010. J. Clin. Microbiol. 48: 3708-3718.

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First Report of Fruit Stem Anthracnose of Kiwifruit Caused by Glomerella septospora in China

Plant Disease ◽

10.1094/pdis-12-12-1173-pdn ◽

2013 ◽

Vol 97 (8) ◽

pp. 1115-1115

Author(s):

C. Li ◽

J. X. Jiang

Keyword(s):

Fruit Set ◽

Southern China ◽

Morphological Characteristics ◽

Its Region ◽

Negative Control ◽

Ethanol Solution ◽

Jiangxi Province ◽

First Report ◽

Pure Cultures ◽

Actinidia Spp

Kiwifruit (Actinidia spp.) is a very economically important fruit crop in southern China, namely Fengxin County. In 2011 and 2012, a new anthracnose on the fruit stem of kiwifruit with blight symptoms was observed during the blossom and fruit-set periods. This caused considerable blossom and fruit drop. To isolate the causal pathogen, symptomatic fruit-stem tissue at the junction of diseased and healthy tissue was cut into 30-mm-long segments. The segments were surface-sterilized in 70% ethanol solution for 10 s, followed by 0.1% mercuric chloride solution for 3 min, and a final set of three rinses in sterile distilled water. Segments were plated onto PDA and incubated at 25°C on a 12-h alternating light-dark cycle (1). Colonies emerging from plant tissues were then transferred to new PDA plates to obtain pure cultures. Colonies grew at the rate of 13.75 mm/day, and were initially white, then became dark green. After 25 days incubation, black and subglobose perithecia appeared at the center of the colonies. Through microscopic examination of perithecia at 400× magnification, club-shaped asci, usually containing 8 ascospores, were found. Ascospores were cylindrical to narrowly fusiform, straight or slightly curved, mostly 3-septate, and 54.5 to 107.5 × 5.0 to 7.5 μm in size. Combining cultural and morphological characteristics with reports in the literature (2), the pathogen was preliminarily identified as a Glomerella species. For six isolates, the rDNA internal transcribed spacer (ITS) region was amplified with the primers ITS1 and ITS4, and the sequences were deposited in GenBank (JX885687). A BLASTn search of GenBank showed that the ITS sequence of our Glomeralla isolate (JX885687) had 100% homology with that of an isolate of Glomeralla septospora (GU935911). Twenty representative isolates (G. septospora) obtained from infected fruit stem tissues in the blossom period and the fruit-set period, respectively, were selected for pathogenicity tests on detached young fruit stems of kiwifruit (JinKui). Each isolate was inoculated into three fruit stems, each of which was slightly wounded with a sterile scalpel and inoculated with a 5-mm-diameter colonized potato dextrose agar (PDA) plug. Negative control stems were treated with the sterile PDA plugs. The inoculated stems were incubated in a growth chamber at 25°C and 95% relatively humidity. After 4 days, characteristic symptoms of fruit stem anthracnose were observed on all inoculated stems, whereas the controls remained asymptomatic. Pure cultures of G. septospora were recovered from all inoculated fruit stems, fulfilling Koch's postulates. These results showed G. septospora is the pathogen of fruit stem anthracnose of kiwifruit, and to our knowledge, this is the first report of G. septospora causing fruit stem anthracnose of kiwifruit in Fengxin county, Jiangxi Province of China, as well as in the world. References: (1) J. Y. Lu. Plant Pathogenic Mycology. China Agriculture Press, Beijing, China, 2004. (2) A. Sivanesan and W. H. Hsieh. Mycological Research, 97:1523, 1993.

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First Report on Neopestalotiopsis aotearoa of Rubber Tree in China

Plant Disease ◽

10.1094/pdis-09-20-1930-pdn ◽

2020 ◽

Author(s):

Boxun Li ◽

Xianbao Liu ◽

Cai Jimiao ◽

Yanli Feng ◽

Guixiu Huang

Keyword(s):

Rubber Tree ◽

Elongation Factor ◽

Morphological Characteristics ◽

Its Region ◽

Raw Material ◽

Conidial Suspension ◽

Basal Cells ◽

Leaf Fall ◽

First Report ◽

Leaf Tissues

Natural rubber is an important industrial raw material and an economically important perennial in China. In recent years, A new leaf fall disease, caused by Neopestalotiopsis aotearoa Maharachch., K.D. Hyde & Crous, has occurred in Indonesia, Malaysia, Thailand, Sri Lanka, and other major rubber planting countries. In May and July of 2020, this disease was first found on 2-year-old rubber seedlings in two plantations located in Ledong and Baisha counties in Hainan Province, China. In the two plantations of approximately 32 ha, 15% of the rubber seedlings had the disease and the defoliation was more than 20%. The infected leaves turned yellow and watery, and dark brown and nearly round lesions of 1-2 mm in diameter were formed on the leaves. When the humidity was high, the center of the lesion was grey-white, and the lesions had many small black dots, black margins and surrounded by yellow halos. When the disease was severe, leaves fell off. To identify the pathogen, leaf tissues were collected from lesion margins after leaf samples were surface-sterilized in 75% ethanol, rinsed with sterile water for three times, and air dried. The leaf tissues were plated on potato dextrose agar (PDA) and incubated at 28°C for seven days. Fungal cultures with similar morphology were isolated from 90% of tested samples and two isolates (HNPeHNLD2001 and HNPeHNLD2002) were used in pathogenicity and molecular tests. Rubber leaves (clone PR107) were inoculated with conidial suspension (106 conidia/ml), and inoculated with PDA were used as the control, Each treatment had 3 leaves, and each leaf was inoculated with 3 spots and incubated at 28oC under high moisture conditions. Five days later, leaves inoculated with conidial suspension showed black leaf spots resembling the disease in the field, whereas the control leaves remained symptomless. The fungal cultures isolated from the inoculated tissues, had identical morphology compared with the initial isolates. Colonies on PDA were 55–60 mm in diameter after seven days at 28°C, with undulate edges, pale brown, thick mycelia on the surface with black, gregarious conidiomata; and the reverse side was similar in color. Black conidia were produced after eight days of culture on PDA. Conidia were fusoid, ellipsoid, straight to slightly curved, 4-septate, ranged from 18.35 to 27.12 μm (mean 22.34 μm) × 4.11 to 7.03 μm (mean 5.41 μm). The basal cells were conic with a truncate base, hyaline, rugose and thin-walled, 4.35 to 6.33 μm long (mean 4.72 μm). Three median cells were doliform, 12.53 to 18.97 μm long (mean 15.26 μm), hyaline, cylindrical to subcylindrical, thin- and smooth-walled, with 2–3 tubular apical appendages, arising from the apical crest, unbranched, filiform, 14.7 to 25.3 μm long (mean 19.94 μm). The basal appendages were singlar, tubular, unbranched, centric, 3.13 to 7.13 μm long (mean 5.48 μm). Morphological characteristics of the isolates were similar to the descriptions of N. aotearoa (Maharachchikumbura et al. 2014). The rDNA internal transcribed spacer (ITS) region, translation elongation factor 1-αgenes (TEF), and beta-tubulin (TUB2) gene were amplified using the primer pairs ITS1/ITS4, EF1-728F/EF1-986R and T1/Bt-2b (Pornsuriya et al. 2020), respectively. The sequences of these genes were deposited in GenBank (ITS Accession Nos.: MT764947 and MT764948; TUB2: MT796262 and MT796263; TEF: MT800516 and MT800517). According to the latest classification of Neoprostalotiopsis spp. (Maharachchikumbura et al. 2014) and multilocus phylogeny, isolates HNPeHNLD2001 and HNPeHNLD2002 were clustered in the same branch with N. aotearoa. Thus, the pathogen was identified as N. aotearoa, which is different from N. cubana and N. formicarum reported in Thailand (Pornsuriya et al. 2020; Thaochan et al. 2020). The Neopestalotiopsis leaf spotdisease of rubber tree (H. brasiliensis) was one of the most serious and destructive leaf diseases in major rubber planting countries in Asia. ( Tajuddin et al. 2020) The present study of leaf fall disease on rubber tree caused byN. aotearoa is the first report in China. The finding provides the basic pathogen information for further monitoring the disease and its control.

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First Report of Black Stem of Avicennia marina Caused by Fusarium equiseti in China

Plant Disease ◽

10.1094/pdis-08-13-0873-pdn ◽

2014 ◽

Vol 98 (6) ◽

pp. 843-843 ◽

Cited By ~ 6

Author(s):

N.-H. Lu ◽

Q.-Z. Huang ◽

H. He ◽

K.-W. Li ◽

Y.-B. Zhang

Keyword(s):

Morphological Characteristics ◽

Its Region ◽

Avicennia Marina ◽

Optical Microscope ◽

Ethanol Solution ◽

Morphological Observation ◽

Fusarium Equiseti ◽

First Report ◽

Initial Symptoms ◽

Β Tubulin

Avicennia marina is a pioneer species of mangroves, a woody plant community that periodically emerges in the intertidal zone of estuarine regions in tropical and subtropical regions. In February 2013, a new disease that caused the stems of A. marina to blacken and die was found in Techeng Island of Zhanjiang, Guangdong Province, China. Initial symptoms of the disease were water-soaked brown spots on the biennial stems that coalesced so whole stems browned, twigs and branches withered, leaves defoliated, and finally trees died. This disease has the potential to threaten the ecology of the local A. marina community. From February to May 2013, 11 symptomatic trees were collected in three locations on the island and the pathogen was isolated as followed: tissues were surface disinfected with 75% ethanol solution (v/v) for 20 s, soaked in 0.1% mercuric chloride solution for 45 s, rinsed with sterilized water three times, dried, placed on potato dextrose agar (PDA), and incubated for 3 to 5 days at 28°C without light. Five isolates (KW1 to KW5) with different morphological characteristics were obtained, and pathogenic tests were done according Koch's postulates. Fresh wounds were made with a sterile needle on healthy biennial stems of A. marina, and mycelial plugs of each isolate were applied and covered with a piece of wet cotton to maintain moisture. All treated plants were incubated at room temperature. Similar symptoms of black stem were observed only on the stems inoculated the isolate KW5 after 35 days, while the control and all stems inoculated with the other isolates remained symptomless. An isolate similar to KW5 was re-isolated from the affected materials. The pathogenic test was repeated three times with the same conditions and it was confirmed that KW5 was the pathogen causing the black stem of A. marina. Hyphal tips of KW5 were transferred to PDA medium in petri dishes for morphological observation. After 48 to 72 h, white, orange, or brown flocculence patches of KW5 mycelium, 5.0 to 6.0 cm in diameter, grew. Tapering and spindle falciform macroconidia (11 to 17.3 μm long × 1.5 to 2.5 μm wide) with an obviously swelled central cell and narrow strips of apical cells and distinctive foot cells were visible under the optical microscope. The conidiogenous cells were intertwined with mycelia and the chlamydospores were globose and formed in clusters. These morphological characteristics of the isolate KW5 are characteristic of Fusarium equiseti (1). For molecular identification, the ITS of ribosomal DNA, β-tubulin, and EF-1α genes were amplified using the ITS4/ITS5 (5), T1/T2 (2), and EF1/EF2 (3) primer pairs. These sequences were deposited in GenBank (KF515650 for the ITS region; KF747330 for β-tubulin region, and KF747331 for EF-1α region) and showed 98 to 99% identity to F. equiseti strains (HQ332532 for ITS region, JX241676 for β-tubulin gene, and GQ505666 for EF-1α region). According to both morphological and sequences analysis, the pathogen of the black stem of A. marina was identified as F. equiseti. Similar symptoms on absorbing rootlets and trunks of A. marina had been reported in central coastal Queensland, but the pathogen was identified as Phytophthora sp. (4). Therefore, the disease reported in this paper differs from that reported in central coastal Queensland. To our knowledge, this is the first report of black stems of A. marina caused by F. equiseti in China. References: (1) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual, 1st ed. Wiley-Blackwell, Hoboken, NJ, 2006. (2) K. O'Donnell and E. Cigelnik. Mol. Phylogenet. Evol. 7:103, 1997. (3) K. O'Donnell et al. Proc. Natl. Acad. Sci. USA. 95:2044, 1998. (4) K. G. Pegg. Aust et al. Plant Pathol. 3:6, 1980. (5) A. W. Zhang et al. Plant Dis. 81:1143, 1997.

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First Report of Fruit Rot of Melon Caused by Fusarium asiaticum in China

Plant Disease ◽

10.1094/pdis-08-20-1857-pdn ◽

2020 ◽

Author(s):

Fangmin Hao ◽

Quanyu Zang ◽

Weihong Ding ◽

Erlei Ma ◽

Yunping Huang ◽

...

Keyword(s):

Disease Incidence ◽

Elongation Factor ◽

Morphological Characteristics ◽

Its Region ◽

Fruit Rot ◽

Post Inoculation ◽

Basal Cells ◽

First Report ◽

Fusarium Asiaticum ◽

Sterile Needle

Melon (Cucumis melo L.) is a member of the Cucurbitaceae family, an important economical and horticultural crop, which is widely grown in China. In May 2020, fruit rot disease with water-soaked lesions and pink molds on cantaloupe melons was observed in several greenhouses with 50% disease incidence in Ningbo, Zhejiang Province in China. In order to know the causal agent, diseased fruits were cut into pieces, surface sterilized for 1 min with 1% sodium hypochlorite (NaClO), 2 min with 75% ethyl alcohol, rinsed in sterile distilled water three times (Zhou et al. 2018), and then placed on potato dextrose agar (PDA) medium amended with streptomycin sulfate (100 μg/ml) plates at 25°C for 4 days. The growing hyphae were transferred to new PDA plates using the hyphal tip method, putative Fusarium colonies were purified by single-sporing. Twenty-five fungal isolates were obtained and formed red colonies with white aerial mycelia at 25°C for 7 days, which were identified as Fusarium isolates based on the morphological characteristics and microscopic examination. The average radial mycelial growth rate of Fusarium isolate Fa-25 was 11.44 mm/day at 25°C in the dark on PDA. Macroconidia were stout with curved apical and basal cells, usually with 4 to 6 septa, and 29.5 to 44.2 × 3.7 to 5.2 μm on Spezieller Nährstoffarmer agar (SNA) medium at 25°C for 10 days (Leslie and Summerell 2006). To identify the species, the internal transcribed spacer (ITS) region and translational elongation factor 1-alpha (TEF1-α) gene of the isolates were amplified and cloned. ITS and TEF1-α was amplified using primers ITS1/ITS4 and EF1/EF2 (O’Donnell et al. 1998), respectively. Sequences of ITS (545 bp, GenBank Accession No. MT811812) and TEF1-α (707 bp, GenBank Acc. No. MT856659) for isolate Fa-25 were 100% and 99.72% identical to those of F. asiaticum strains MSBL-4 (ITS, GenBank Acc. MT322117.1) and Daya350-3 (TEF1-α, GenBank Acc. KT380124.1) in GenBank, respectively. A phylogenetic tree was established based on the TEF1-α sequences of Fa-25 and other Fusarium spp., and Fa-25 was clustered with F. asiaticum. Thus, both morphological and molecular characterizations supported the isolate as F. asiaticum. To confirm the pathogenicity, mycelium agar plugs (6 mm in diameter) removed from the colony margin of a 2-day-old culture of strain Fa-25 were used to inoculate melon fruits. Before inoculation, healthy melon fruits were selected, soaked in 2% NaClO solution for 2 min, and washed in sterile water. After wounding the melon fruits with a sterile needle, the fruits were inoculated by placing mycelium agar plugs on the wounds, and mock inoculation with mycelium-free PDA plugs was used as control. Five fruits were used in each treatment. The inoculated and mock-inoculated fruits were incubated at 25°C with high relative humidity. Symptoms were observed on all inoculated melon fruits 10 days post inoculation, which were similar to those naturally infected fruits, whereas the mock-inoculated fruits remained symptomless. The fungus re-isolated from the diseased fruits resembled colony morphology of the original isolate. The experiment was conducted three times and produced the same results. To our knowledge, this is the first report of fruit rot of melon caused by F. asiaticum in China.

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First Report of Leaf Blight Caused by Septoria allii on Garlic Chives in Korea

Plant Disease ◽

10.1094/pdis-08-12-0809-pdn ◽

2013 ◽

Vol 97 (1) ◽

pp. 147-147

Author(s):

J. H. Park ◽

S. E. Cho ◽

K. S. Han ◽

H. D. Shin

Keyword(s):

Disease Incidence ◽

Morphological Characteristics ◽

Its Region ◽

Leaf Blight ◽

Plant Diseases ◽

Cultivated Plants ◽

Sequence Information ◽

Conidial Suspension ◽

Korean Society ◽

First Report

Garlic chives, Allium tuberosum Roth., are widely cultivated in Asia and are the fourth most important Allium crop in Korea. In June 2011, a leaf blight of garlic chives associated with a Septoria spp. was observed on an organic farm in Hongcheon County, Korea. Similar symptoms were also found in fields within Samcheok City and Yangku County of Korea during the 2011 and 2012 seasons. Disease incidence (percentage of plants affected) was 5 to 10% in organic farms surveyed. Diseased voucher specimens (n = 5) were deposited at the Korea University Herbarium (KUS). The disease first appeared as yellowish specks on leaves, expanding to cause a leaf tip dieback. Half of the leaves may be diseased within a week, especially during wet weather. Pycnidia were directly observed in leaf lesions. Pycnidia were amphigenous, but mostly epigenous, scattered, dark brown to rusty brown, globose, embedded in host tissue or partly erumpent, separate, unilocular, 50 to 150 μm in diameter, with ostioles of 20 to 40 μm in diameter. Conidia were acicular, straight to sub-straight, truncate at the base, obtuse at the apex, hyaline, aguttulate, 22 to 44 × 1.8 to 3 μm, mostly 3-septate, occasionally 1- or 2-septate. These morphological characteristics matched those of Septoria allii Moesz, which is differentiated from S. alliacea on conidial dimensions (50 to 60 μm long) (1,2). A monoconidial isolate was cultured on potato dextrose agar (PDA). Two isolates have been deposited in the Korean Agricultural Culture Collection (Accession Nos. KACC46119 and 46688). Genomic DNA was extracted using the DNeasy Plant Mini DNA Extraction Kit (Qiagen Inc., Valencia, CA). The internal transcribed spacer (ITS) region of rDNA was amplified using the ITS1/ITS4 primers and sequenced. The resulting sequence of 482-bp was deposited in GenBank (JX531648 and JX531649). ITS sequence information was at least 99% similar to those of many Septoria species, however no information was available for S. allii. Pathogenicity was tested by spraying leaves of three potted young plants with a conidial suspension (2 × 105 conidia/ml), which was harvested from a 4-week-old culture on PDA. Control leaves were sprayed with sterile water. The plants were placed in humid chambers (relative humidity 100%) for the first 48 h. After 7 days, typical leaf blight symptoms started to develop on the leaves of inoculated plants. S. allii was reisolated from the lesions of inoculated plants, confirming Koch's postulates. No symptoms were observed on control plants. The host-parasite association of A. tuberosum and S. allii has been known only from China (1). S. alliacea has been recorded on several species of Allium, e.g. A. cepa, A. chinense, A. fistulosum, and A. tuberosum from Japan (4) and A. cepa from Korea (3). To the best of our knowledge, this is the first report of S. allii on garlic chives. No diseased plants were observed in commercial fields of garlic chives which involved regular application of fungicides. The disease therefore seems to be limited to organic garlic chive production. References: (1) P. K. Chi et al. Fungous Diseases on Cultivated Plants of Jilin Province, Science Press, Beijing, China, 1966. (2) P. A. Saccardo. Sylloge Fungorum Omnium Hucusque Congnitorum. XXV. Berlin, 1931. (3) The Korean Society of Plant Pathology. List of Plant Diseases in Korea, Suwon, Korea, 2009. (4) The Phytopathological Society of Japan. Common Names of Plant Diseases in Japan, Tokyo, Japan, 2000.

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First Report of Nigrospora Leaf Blight on Tea Caused by Nigrospora sphaerica in India

Plant Disease ◽

10.1094/pdis-05-14-0545-pdn ◽

2015 ◽

Vol 99 (3) ◽

pp. 417-417 ◽

Cited By ~ 11

Author(s):

J. Dutta ◽

S. Gupta ◽

D. Thakur ◽

P. J. Handique

Keyword(s):

Camellia Sinensis ◽

West Bengal ◽

Morphological Characteristics ◽

Its Region ◽

Causative Organism ◽

Distilled Water ◽

Conidial Suspension ◽

Tea Leaves ◽

First Report ◽

Initial Symptoms

Tea [Camellia sinensis (L.) O. Kuntze] is an economically important non-alcoholic caffeine-containing beverage crop widely cultivated for leaves in India, especially in the Darjeeling district of West Bengal. In May 2012, distinct blight symptoms were observed on leaves of popular tea cultivars AV-2, Tukdah 78, Rungli Rungliot 17/144, and Bannockburn 157 in commercial tea estates of the Darjeeling district. This disease reduces yield and quality of the leaves. The initial symptoms were frequently observed on the young leaf margins and apices. Foliar symptoms are characterized by grayish to brown, semicircular or irregular shaped lesions, often surrounded by pale yellow zones up to 9 mm in diameter. The lesions later expand and the affected leaves turn grayish to dark brown and eventually the dried tissue falls, leading to complete defoliation of the plant. The disease causes damage to leaves of all ages and is severe in young leaves. A portion of the symptomatic leaf tissues were surface sterilized in 70% ethanol for 30 s, then in 2% NaClO for 3 min, rinsed three times in sterile distilled water, and plated onto potato dextrose agar (PDA). The fungal colonies were initially white and then became grayish to brown with sporulation. Conidia were spherical to sub spherical, single-celled, black, 19 to 21 μm in diameter, and were borne on a hyaline vesicle at the tip of each conidiophore. Morphological characteristics of the isolates were concurring to those of Nigrospora sphaerica (1). Moreover, the internal transcribed spacer (ITS) region of the ribosomal RNA was amplified by using primers ITS1 and ITS4 and sequenced (GenBank Accession No. KJ767520). The sequence was compared to the GenBank database through nucleotide BLAST search and the isolate showed 100% similarity to N. sphaerica (KC519729.1). On the basis of morphological characteristics and nucleotide homology, the isolate was identified as N. sphaerica. Koch's postulates were fulfilled in the laboratory on tea leaves inoculated with N. sphaerica conidial suspension (106 conidia ml−1) collected from a 7-day-old culture on PDA. Six inoculated 8-month-old seedlings of tea cultivars AV-2 and S.3/3 were incubated in a controlled environment chamber at 25°C and 80 to 85% humidity with a 12-h photoperiod. In addition, three plants of each cultivar were sprayed with sterile distilled water to serve as controls. Twelve to 14 days after inoculation, inoculated leaves developed blight symptoms similar to those observed on naturally infected tea leaves in the field. No symptoms were observed on the control leaves. The pathogen was re-isolated from lesions and its identity was confirmed by morphological characteristics. It was reported that N. sphaerica is frequently encountered as a secondary invader or as a saprophyte on many plant species and also as a causative organism of foliar disease on several hosts worldwide (2,3). To our knowledge, this is first report of N. sphaerica as a foliar pathogen of Camellia sinensis in Darjeeling, West Bengal, India, or worldwide. References: (1) M. B. Ellis. Dematiaceous Hyphomycetes. CMI, Kew, Surrey, UK, 1971. (2) D. F. Farr and A. Y. Rossman. Fungal Databases, Syst. Mycol. Microbiol. Lab., ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ July 01, 2013. (3) E. R. Wright et al. Plant Dis. 92:171, 2008.

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First Report of Postharvest Blue Mold decay caused by Penicillium expansum on Lemon (Citrus limon) fruit in China

Plant Disease ◽

10.1094/pdis-10-20-2239-pdn ◽

2021 ◽

Author(s):

Ibatsam Khokhar ◽

Jianming Chen ◽

Junhuan Wang ◽

Yang Jia ◽

Yanchun Yan ◽

...

Keyword(s):

Morphological Characteristics ◽

Its Region ◽

Penicillium Expansum ◽

Citrus Limon ◽

Conidial Suspension ◽

Blue Mold ◽

Causal Organism ◽

Lemon Fruit ◽

First Report ◽

Fungal Isolates

Lemon (Citrus limon) is one of the most important commercial (both dried and fresh) citrus fruits in China. In the spring of 2019, postharvest blue mold decay was observed at an incidence of 3-5% on lemon fruit at the local markets in Beijing, China. Fruit lesions were circular, brown, soft, and watery, and rapidly expanded at 25°C. To isolate the causal organism, small pieces (2 mm3) were cut from the lesions, surface-sterilized for 1 min in 1.5% NaOCl, rinsed three times with sterilized water, dried with sterile filter paper, placed onto potato dextrose agar (PDA) medium, and incubated at 25°C for 6 days. Eight morphologically similar single-colony fungal isolates were recovered from six lemon fruit. Colony surfaces were bluish-green on the upper surface and cream to yellow-brown one the reverse. Hyphae on colony margins were entirely subsurface and cream in color. Mycelium was highly branched, septate, and colorless, and conidiophores were 250 to 450 × 3.0 to 4.0 µm in size. Stipe of conidiophores were smooth-walled, bearing terminal penicilli, typically terverticillate or less commonly birverticillate, rami occurring singly, 16 to 23 × 3.0 to 4.0 µm, metulae in 3 to 6, measuring 12 to 15 × 3.0 to 4.0 µm. Phialides were ampulliform to almost cylindrical, in verticils of 5 to 8, measuring 8 to 11 × 2.5 to 3.2 µm with collula. Conidia were smooth-walled, ellipsoidal, measuring 3.0 to 3.5 × 2.5 to 3.0 µm. According to morphological characteristics, the fungus was identified as Penicillium expansum (Visagie et al. 2014). For molecular identification, genomic DNA of eight fungal isolates was extracted, regions of the beta-tubulin (TUB), and calmodulin (CAL) genes and ITS region, were amplified using Bt2a/Bt2b, CAL-228F/ CAL-737, and ITS1/ITS4 primers respectively. Obtained sequences of all isolates were identical to sequences of the representative isolate YC-IK12, which was submitted in the GenBank. BLAST results of YC-IK12 sequences (ITS; MT856700: TUB; MT856958: CAL; MT856959) showed 98 to 100% similarity with P. expansum accessions (NR-077154, LN896428, JX141581). For pathogenicity tests, 10 μl of conidial suspension (10 × 105 conidia/ml) from seven-day-old YC-IK12 culture was inoculated using a sterilized needle into the surface of each five asymptomatic disinfected lemons. As a control, three lemons were inoculated using sterile distilled water. All inoculated lemons were placed in plastic containers and incubated at 25°C for 7 days. Decay lesions, identical to the original observations, developed on all inoculated lemons, while control lemons remained asymptomatic. Fungus re-isolated from the inoculated lemon was identified as P. expansum on the basis morphology and Bt2a/Bt2b, CAL-228F/ CAL-737, and ITS1/ITS4 sequences. Previously, Penicillium spp. including P. expansum have been reported as post-harvest pathogens on various Citrus spp. (Louw & Korsten 2015). However, P. digitatum has been reported on lemons and P. expansum has been reported on stored Kiwifruit (Actinidia arguta), Malus, and Pyrus species in China (Tai, 1979; Wang et al. 2015). To our knowledge, this is the first report of blue mold caused by P. expansum on lemons in China. References Louw, J. P., Korsten, L. 2015. Plant Dis. 99:21-30. Tai, F.L. 1979. Sylloge Fungorum Sinicorum. Sci. Press, Acad. Sin., Peking, 1527 pages. 8097 Visagie, C.M. et al. 2014. Studies. Mycol.78: 343. Wang, C. W. et al. 2015. Plant Dis. 99:1037.

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First report of fruit blight caused by Alternaria alternata on sesame in Northeast China

Plant Disease ◽

10.1094/pdis-01-21-0047-pdn ◽

2021 ◽

Author(s):

Hongsen Cheng ◽

De Xue Gao ◽

Huijie Sun ◽

Yanbin Na ◽

Jing Xu

Keyword(s):

Alternaria Alternata ◽

Morphological Characteristics ◽

Its Region ◽

Liaoning Province ◽

Oilseed Crop ◽

Distilled Water ◽

Conidial Suspension ◽

First Report ◽

Health Products ◽

Blight Disease

Sesame (Sesamum indicum L.) is an important oilseed crop in China and it is also used in food and health products. In August of 2019, a blight sesame fruit was observed in a field of Liaoyang city, Liaoning province of China. Initial disease symptoms consisted of brown or dark brown spots on fruit. With time, lesions coalesced and the whole fruit turned dark brown or black. Most of the diseased fruit had thin and small, deformed, necrotic, hardened cracked epidermal lesions. Lesions were also produced on stem and petioles leading to leaf abscission. The disease results in premature fruit death, and in turn, considerable yield losses. To determine the causal agent, symptomatic fruit with developing lesions were collected, and surface sterilized in 2% NaClO for 3 min, rinsed three times in distilled water, and plated onto PDA medium. After incubation at 25°C for 5 days, a dark olivaceous fungus with abundant, branched, brown to black, and septate hyphae was consistently isolated. Twenty single spores were separated with an inoculation needle under stereomicroscope. The conidia were in chains, brown, obclavate, ovoid or ellipsoid, with 1-6 transverse septa and 0-4 longitudinal or oblique septa 12.5 to 45 × 6.5 to 14.5 μm in size. Conidiophores were septate, light brown to olive brown, measuring 22-60 μm × 2-4 μm. The morphological characteristics of the 20 isolates all matched the description of Alternaria alternata (Simmons, 2007). The internal transcribed spacer (ITS) region of rDNA of 15 isolates was amplified using primers ITS1/ITS4 (White et al. 1990) and EF1-728F/EF1-986R (Carbone et al. 1999) and sequenced. Identical sequences were obtained and the sequence of the isolate ZMHG12 was submitted to GenBank (Accession no. MW418181 and MW700316). BLAST analysis of the sequences of the isolates of ZMHG12 showed 100% to A. alternata (KP739875 and LC132712). In pathogenicity tests, a conidial suspension (2.5 × 105 conidia per ml) was prepared from 7 days-old cultures of isolate ZMHG12 grown on PDA at 25°C. Fruit of 10 two-month-old potted sesame plants (Variety “Liaozhi 8”) were sprayed with the conidia suspension until runoff. Another 10 plants sprayed with distilled water to served as non-inoculated controls. All plants were maintained for 48 h in a humid chamber with a temperature of 25°C to 26°C, and then moved to a greenhouse. Ten days after inoculation, all fruit of inoculated plants exhibited symptoms similar to those observed in the field and non-inoculated control plants remained symptomless. The experiment was repeated twice with similar results. A. alternata has been reported as a pathogen caused leaf blight disease of sesame in Pakistan (Nayyar et al. 2017). To our knowledge, this is the first report of A.alternata causing fruit blight of sesame in China. To date, we have observed the disease on sesames in fields of Fuxin, Chaoyang and Tieling city in Liaoning Province, and Tongliao city in Inner Mongolia of China, and it has become an important disease in sesame production of China. References : Simmons E. G. 2007. Alternaria: An identification manual. CBS Fungal Biodiversity Center, Utrecht, Netherlands. White T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego. Carbone I., et al. 1999. Mycologia, 91: 553-556. Nayyar, B. G., et al. 2017. Plant Pathology Journal, 33 (6): 543-553.

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