First report of Fusarium incarnatum causing fruit rot of litchi in China

Litchi (Litchi chinensis Sonn.) is an indigenous tropical and subtropical fruit in Southern China with an attractive appearance, delicious taste, and good nutritional value (Jiang et al. 2003). In March 2020, brown rots were observed on nearly ripe litchi fruits (cv. Guihuaxiang) in an orchard of Lingshui county, Hainan province of China (18.615877° N, 109.948871° E). About 5% fruits were symptomatic in the field, and the disease caused postharvest losses during storage. The initial infected fruits had no obvious symptoms on the outer pericarp surfaces, but appeared irregular, brown to black-brown lesions in the inner pericarps around the pedicels. Then lesions expanded and became brown rots. Small tissues (4 mm × 4 mm) of fruit pericarps were cut from symptomatic fruits, surface-sterilized in 1% sodium hypochlorite for 3 min, rinsed in sterilized water three times, plated on potato dextrose agar (PDA) and incubated at 28℃ in the darkness. Morphologically similar colonies were isolated from 85% of 20 samples after 4 days of incubation. Ten isolates were purified using a single-spore isolation method. The isolates grown on PDA had abundant, fluffy, whitish to yellowish aerial mycelia, and the reverse side of the Petri dish was pale brown. Morphological characteristics of conidia were further determined on carnation leaf-piece agar (CLA) (Leslie et al. 2006). Macroconidia were straight to slightly curved, 3- to 5-septates with a foot-shaped basal cell, tapered at the apex, 2.70 to 4.43 µm × 18.63 to 37.58 µm (3.56 ± 0.36 × 28.68 ± 4.34 µm) (n = 100). Microconidia were fusoid to ovoid, 0- to 1-septate, 2.10 to 3.57 µm × 8.18 to 18.20 µm (2.88 ± 0.34 × 11.71 ± 1.97 µm) (n = 100). Chlamydospores on hyphae singly or in chains were globose, subglobose, or ellipsoidal. Based on cultural features and morphological characteristics, the fungus was identified as a Fusarium species (Leslie et al. 2006). To further confirm the pathogen, DNA was extracted from the 7-day-old aerial mycelia of three isolates (LZ-1, LZ-3, and LZ-5) following Chohan et al. (2019). The sequences of the internal transcribed spacer region of rDNA (ITS), translation elongation factor-1 alpha (tef1) gene, and histone H3 (his3) gene were partially amplified using primers ITS1/ITS4, EF1-728F/EF1-986R, and CYLH3F/CYLH3R, respectively (Funnell-Harris et al. 2017). The nucleotide sequences were deposited in GenBank (ITS: 515 bp, MW029882, 533 bp, MW092186, and 465 bp, MW092187; tef1: 292 bp, MW034437, 262 bp, MW159143, and 292 bp, MW159141; his3: 489 bp, MW034438, 477 bp, MW159142, and 474 bp, MW159140). The ITS, tef1, and his3 genes showed 99-100% similarity with the ITS (MH979697), tef1 (MH979698), and his3 (MH979696) genes, respectively of Fusarium incarnatum (TG0520) from muskmelon fruit. The phylogenetic analysis of the tef1 and his3 gene sequences showed that the three isolates clustered with F. incarnatum. Pathogenicity tests were conducted by spraying conidial suspension (1×106 conidia/ml) on wounded young fruits in the orchid. Negative controls were sprayed with sterilized water. Fruits were bagged with polythene bags for 24 hours and then unbagged for 10 days. Each treatment had 30 fruits. The inoculated fruits developed symptoms similar to those observed in the orchard and showed light brown lesions on the outer pericarp surfaces and irregular, brown to black-brown lesions in the inner pericarps, while the fruits of negative control remained symptomless. The same fungus was successfully recovered from symptomatic fruits, and thus, the test for the Koch’s postulates was completed. F. semitectum (synonym: F. incarnatum) (Saha et al. 2005), F. oxysporum (Bashar et al. 2012), and F. moniliforme (Rashid et al. 2015) have been previously reported as pathogens causing litchi fruit rots in India and Bangladesh. To our knowledge, this is the first report of Fusarium incarnatum causing litchi fruit rot in China.

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First Report of Leaf Spot Caused by Botrytis cinerea on Cardamine hupingshanensis in China

Plant Disease ◽

10.1094/pdis-04-21-0698-pdn ◽

2021 ◽

Author(s):

Jun Guo ◽

Jin Chen ◽

Zhao Hu ◽

Jie Zhong ◽

Jun Zi Zhu

Keyword(s):

Botrytis Cinerea ◽

Morphological Characteristics ◽

Gray Mold ◽

Hunan Province ◽

Conidial Suspension ◽

Heat Shock Protein 60 ◽

Basal Cells ◽

Single Spore ◽

Internal Transcribed Spacer Region ◽

First Report

Cardamine hupingshanensis is a selenium (Se) and cadmium (Cd) hyperaccumulator plant distributed in wetlands along the Wuling Mountains of China (Zhou et al. 2018). In March of 2020, a disease with symptoms similar to gray mold was observed on leaves of C. hupingshanensis in a nursery located in Changsha, Hunan Province, China. Almost 40% of the C. hupingshanensis (200 plants) were infected. Initially, small spots were scattered across the leaf surface or margin. As disease progressed, small spots enlarged to dark brown lesions, with green-gray, conidia containing mold layer under humid conditions. Small leaf pieces were cut from the lesion margins and were sterilized with 70% ethanol for 10 s, 2% NaOCl for 2 min, rinsed with sterilized distilled water for three times, and then placed on potato dextrose agar (PDA) medium at 22°C in the dark. Seven similar colonies were consistently isolated from seven samples and further purified by single-spore isolation. Strains cultured on PDA were initially white, forming gray-white aerial mycelia, then turned gray and produced sclerotia after incubation for 2 weeks, which were brown to blackish, irregular, 0.8 to 3.0 × 1.2 to 3.5 mm (n=50). Conidia were unicellular, globose or oval, colourless, 7.5 to 12.0 × 5.5 to 8.3 μm (n=50). Conidiophores arose singly or in group, straight or flexuous, septate, brownish to light brown, with enlarged basal cells, 12.5 to 22.1 × 120.7 to 310.3 μm. Based on their morphological characteristics in culture, the isolates were putatively identified as Botrytis cinerea (Ellis 1971). Genomic DNA of four representative isolates, HNSMJ-1 to HNSMJ-4, were extracted by CTAB method. The internal transcribed spacer region (ITS), glyceraldehyde-3-phosphate dehydrogenase gene (G3PDH), heat-shock protein 60 gene (HSP60), ATP-dependent RNA helicaseDBP7 gene (MS547) and DNA-dependent RNA polymerase subunit II gene (RPB2) were amplified and sequenced using the primers described previously (Aktaruzzaman et al. 2018) (MW820311, MW831620, MW831628, MW831623 and MW831629 for HNSMJ-1; MW314722, MW316616, MW316617, MW316618 and MW316619 for HNSMJ-2; MW820519, MW831621, MW831627, MW831624 and MW831631 for HNSMJ-3; MW820601, MW831622, MW831626, MW831625 and MW831630 for HNSMJ-4). BLAST searches showed 99.43 to 99.90% identity to the corresponding sequences of B. cinerea strains, such as HJ-5 (MF426032.1, MN448500.1, MK791187.1, MH727700.1 and KX867998.1). A combined phylogenetic tree using the ITS, G3PDH, HSP60 and RPB2 sequences was constructed by neighbor-joining method in MEGA 6. It revealed that HNSMJ-1 to HNSMJ-4 clustered in the B. cinerea clade. Pathogenicity tests were performed on healthy pot-grown C. hupingshanensis plants. Leaves were surface-sterilized and sprayed with conidial suspension (106 conidia/ mL), with sterile water served as controls. All plants were kept in growth chamber with 85% humidity at 25℃ following a 16 h day-8 h night cycle. The experiment was repeated twice, with each three replications. After 4 to 7 days, symptoms similar to those observed in the field developed on the inoculated leaves, whereas controls remained healthy. The pathogen was reisolated from symptomatic tissues and identified using molecular methods, confirming Koch’s postulates. B. cinerea has already been reported from China on C. lyrate (Zhang 2006), a different species of C. hupingshanensis. To the best of our knowledge, this is the first report of B. cinerea causing gray mold on C. hupingshanensis in China and worldwide. Based on the widespread damage in the nursery, appropriate control strategies should be adopted. This study provides a basis for studying the epidemic and management of the disease.

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First Report of Stemphylium solani as the Causal Agent of a Leaf Spot on Greenhouse Cucumber

Plant Disease ◽

10.1094/pdis-08-12-0776-pdn ◽

2013 ◽

Vol 97 (2) ◽

pp. 287-287 ◽

Cited By ~ 5

Author(s):

D. J. Vakalounakis ◽

E. A. Markakis

Keyword(s):

Leaf Spot ◽

Natural Infection ◽

Apical Cell ◽

Morphological Characteristics ◽

Leaf Spot Disease ◽

Pathogenicity Test ◽

Conidial Suspension ◽

Single Spore ◽

Internal Transcribed Spacer Region ◽

First Report

During the 2011 to 2012 crop season, a severe leaf spot disease of cucumber (Cucumis sativus) cv. Cadiz was noticed on crops in some greenhouses in the Goudouras area, Lasithi, Crete, Greece. Symptoms appeared in late winter, mainly on the leaves of the middle and upper part of the plants. Initially, small necrotic pinpoint lesions with white centers, surrounded by chlorotic halos, 1 to 3 mm in diameter, appeared on the upper leaf surfaces, and these progressively enlarged to spots that could coalesce to form nearly circular lesions up to 2 cm or more in diameter. Stemphylium-like fructifications appeared on necrotic tissue of older lesions. Severely affected leaves became chlorotic and died. No other part of the plant was affected. Small tissue pieces from the edges of lesions were surface disinfected in 0.5% NaClO for 5 min, rinsed in sterile distilled water, plated on acidified potato dextrose agar and incubated at 22 ± 0.5°C with a 12-h photoperiod. Stemphylium sp. was consistently isolated from diseased samples. Colonies showed a typical septate mycelium with the young hyphae subhyaline and gradually became greyish green to dark brown with age. Conidiophores were subhyaline to light brown, 3- to 10-septate, up to 200 μm in length, and 4 to 7 μm in width, with apical cell slightly to distinctly swollen, bearing a single spore at the apex. Conidia were muriform, mostly oblong to ovoid, but occasionally nearly globose, subhyline to variant shades of brown, mostly constricted at the median septum, 22.6 ± 6.22 (11.9 to 36.9) μm in length, and 15.1 ± 2.85 (8.3 to 22.6) μm in width, with 1 to 8 transverse and 0 to 5 longitudinal septa. DNA from a representative single-spore isolate was extracted and the internal transcribed spacer region (ITS) of ribosomal DNA (rDNA) was amplified using the universal primers ITS5 and ITS4. The PCR product was sequenced and deposited in GenBank (Accession No. JX481911). On the basis of morphological characteristics (3) and a BLAST search with 100% identity to the published ITS sequence of a S. solani isolate in GenBank (EF0767501), the fungus was identified as S. solani. Pathogenicity tests were performed by spraying a conidial suspension (105 conidia ml–1) on healthy cucumber (cv. Knossos), melon (C. melo, cv. Galia), watermelon (Citrullus lanatus cv. Crimson sweet), pumpkin (Cucurbita pepo, cv. Rigas), and sponge gourd (Luffa aegyptiaca, local variety) plants, at the 5-true-leaf stage. Disease symptoms appeared on cucumber and melon only, which were similar to those observed under natural infection conditions on cucumber. S. solani was consistently reisolated from artificially infected cucumber and melon tissues, thus confirming Koch's postulates. The pathogenicity test was repeated with similar results. In 1918, a report of a Stemphylium leaf spot of cucumber in Indiana and Ohio was attributed to Stemphylium cucurbitacearum Osner (4), but that pathogen has since been reclassified as Leandria momordicae Rangel (2). That disease was later reported from Florida (1) and net spot was suggested as a common name for that disease. For the disease reported here, we suggest the name Stemphylium leaf spot. This is the first report of a disease of cucumber caused by a species of Stemphylium. References: (1) C. H. Blazquez. Plant Dis. 67:534, 1983. (2) P. Holliday. Page 243 in: A Dictionary of Plant Pathology. Cambridge University Press, Cambridge, UK, 1998. (3) B. S. Kim et al. Plant Pathol. J. 15:348, 1999. (4) G. A. Osner. J. Agric. Res. 13:295, 1918.

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First Report of Maize Stalk Rot Caused by Fusarium nelsonii in China

Plant Disease ◽

10.1094/pdis-01-21-0089-pdn ◽

2021 ◽

Author(s):

Xiaojie Zhang ◽

Cheng Guo ◽

Chunming Wang ◽

Tianwang Zhou

Keyword(s):

Fusarium Species ◽

Morphological Characteristics ◽

Molecular Characteristics ◽

Conidial Suspension ◽

Basal Cells ◽

Sterile Water ◽

Single Spore ◽

First Report ◽

Stalk Rot ◽

Maize Plants

Maize (Zea Mays L.) is one of the main crops in Ningxia Province, China, and stalk rot has become a serious disease of maize in this area. Infected plants showed softening of the stalks at lower internodes, which lodged easily and died prematurely during grain filling, and the pith tissue internally appeared to be disintegrating and slightly brown to reddish. In September 2018, symptomatic tissue was collected from seventeen locations in Ningxia. The incidence ranged from 5% to 40% in surveyed fields, reaching as high as 86% in certain plots. The discolored stalk pith tissues from the lesion region were cut into small pieces (approximately 0.5 × 0.2 cm), superficially disinfected with 75% ethanol for 1 min and rinsed three times with sterile water before plating on potato dextrose agar (PDA) medium with chloromycetin. The purified strains were obtained by single-spore separation and transferred to PDA and carnation leaf agar (CLA) medium. Morphological and molecular characteristics confirmed the presence of nine Fusarium species in these samples, including Fusarium graminearum species complex and Fusarium verticillioides. Four isolates of Fusarium nelsonii were recovered from samples collected in Shizuishan and Wuzhong. On PDA plates, the floccose to powdery, white to rose-colored aerial mycelia were produced and covered plates after 8 days of incubation, producing abundant mesoconidia and chlamydospores. Mesoconidia were fusiform or lanceolate until slightly curved with 0-3 septa, and chlamydospores were initially smooth and transparent, and became verrucous and light brown. Macroconidia produced in CLA were straight or curved and falcate, usually having 3-5 septa, with beak-shaped strongly curved apical cells and foot-shaped basal cells. Two isolates (SS-1-7 and ZY-2-2) were selected for molecular identification, and the total DNA was extracted using a fungal genomic DNA separation kit (Sangon Biotechnology, Shanghai, China). Sequence comparison of EF-1α (GenBank accession numbers MW294197 and MW294198) and RPB2 (Accession MW294176 and MW294177) genes showed 97% homology with the sequences of F. nelsonii reported in GenBank (accession MN120760 for TEF and accession MN120740 for RPB2). Pathogenicity tests with two isolates (SS-1-7 and ZY-2-2) were performed by individually inoculating five 10-leaf stage maize plants at between the 2nd and 3rd stem nodes from the soil level with 20 μl conidial suspension at a concentration of 106 conidia/ml as described by Zhang et al. (2016). Five maize plants inoculated with sterile water were used as controls. The inoculated plants were kept at 25 ± 0.5°C in the greenhouse with a photoperiod of 12 h. After 30 days, all plants inoculated with the conidial suspension formed an internal dark brown necrotic area around the inoculation site, whereas the control plants showed no symptoms. The pathogen was re-isolated from the necrotic tissue of the inoculated plants and identified by morphological characteristics as F. nelsonii. This species was first described by Marasas et al. (1998), and it is expanding its host range and has been isolated from sorghum, Medicago, wheat, and cucumber (Ahmad et al. 2020). The pathogen should be paid more attention owing to a serious risk of trichothecene and aflatoxin contamination (Astoreca et al. 2019; Lincy et al. 2011). To our knowledge, this is the first report of maize stalk rot caused by F. nelsonii in China. References: Ahmad, A., et al. 2020. Plant disease.1542 https://doi.org/10.1094/PDIS-11-19-2511-PDN Astoreca, A. L., et al. 2019. Eur. J. Plant Pathol. 155:381. Lincy, S. V., et al. 2011. World J. Microbiol. Biotechnol. 27:981. Marasas, W. F. O., et al. 1998. Mycologia 90:505. Zhang, Y., et al. 2016. PLoS Pathog. 12:e1005485. Funding: This research was financially supported by National R & D Plan of China (No.2019QZKK0303); Ningxia Agriculture and Forestry Academy Science and Technology Cooperation Project (DW-X-2018019)

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First report of postharvest fruit rot on passion fruit (Passiflora edulis) caused by Lasiodiplodia theobromae in Mainland China

Plant Disease ◽

10.1094/pdis-06-20-1346-pdn ◽

2020 ◽

Author(s):

Wu Zhang ◽

Xue Li Niu ◽

Jin Yu Yang

Keyword(s):

Southern China ◽

Morphological Characteristics ◽

Its Region ◽

Passion Fruit ◽

Fruit Rot ◽

Pathogenicity Test ◽

Single Spore ◽

Passiflora Edulis ◽

Lasiodiplodia Theobromae ◽

First Report

As an economically important tropical and subtropical fruit crop, passion fruit (Passiflora edulis Sims) is widely planted in many provinces of southern China. In April 2017, postharvest fruit rot was observed on 15% to 25% of passion fruit in several fruit markets of Zhanjiang City in Guangdong Province. Initial disease symptoms on infected fruit were irregular, brown, water-soaked lesions, which enlarged into large black and sunken patches. Lesions were usually covered with an abundance of little black dots (pycnidia) and black-gray hyphae. For the pathogen isolation, fifteen symptomatic fruit were randomly collected from three local markets. Fourteen single-spore fungal isolates with similar morphology ware isolated from the infected tissues. Two isolates (ZW 49-1 and ZW 50-1) were randomly selected to further study. The colonies on PDA were initially greyish-white and became dark-gray with age. Abundant globular and irregular pycnidia were observed after incubation at 25 °C for 3 weeks. The conidia of the fungus were initially hyaline, unicellular, apex rounded, thick-walled, and ellipsoid, becoming dark brown, bicellular with longitudinal striations at maturity, 26.4 ± 2.5 × 13.4 ± 1.2 μm (n = 50). The morphology of the fungus resembled Lasiodiplodia theobromae (Pat.) Griff. & Maubl. (Phillips et al. 2013). To confirm species identification, the partial internal transcribed spacer (ITS) region of rDNA, translation elongation factor-alpha (EF1-α) and β-tubulin (TUB) gene were amplified from genomic DNA of the two isolates with the ITS1/ITS4, EF1-688F/EF1-986R, and Bt2a/Bt2b primers, respectively (Glass and Donaldson 1995; Alves et al. 2008; White et al. 1990). Base on the BLASTn analysis, the ITS (MT644473, MT644474), EF1-α (MT649210, MT649211) and TUB (MT649212, MT649213) sequences of both isolates were 100%, 99% and 100% similarity to the L. theobromae CBS 164.96 ex-type sequences in the NCBI database (AY640255, AY640258, and KU887532, respectively) (Phillips et al. 2013). For pathogenicity test, asymptomatic passion fruit were previously disinfested in 0.5 % sodium hypochlorite and superficially wounded with a sterile needle. Five-mm-diameter plugs with mycelial taken from 5-day-old PDA colonies were placed on the wounds. Sterile PDA plugs were used as negative controls. Each treatment had five replicates and the test was repeated twice. Fruit were maintained in plastic boxes to keep at 25°C for one week. One week after inoculation, gray mycelia had covered a majority of the fruit surface and caused a black, sunken rot. The inoculated fungus was reisolated and confirmed as L. theobromae by morphological characteristics. The mock inoculated fruit remained asymptomatic. The occurrence of fruit rot on passion fruit caused by L. theobromae was reported in Taiwan, China recently (Huang et al., 2019). To our knowledge, this is the first report of L. theobromae causing postharvest fruit rot on passion fruit in the Chinese mainland.

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First Report of Apiospora mold on sugarcane in China caused by Apiospora arundinis (Arthrinium arundinis)

Plant Disease ◽

10.1094/pdis-02-21-0386-pdn ◽

2021 ◽

Author(s):

Jie Liao ◽

Wenyan Jiang ◽

Xiaojian Wu ◽

Jie He ◽

Huiling Li ◽

...

Keyword(s):

Southern China ◽

Morphological Characteristics ◽

Saccharum Officinarum ◽

Causal Agent ◽

Conidial Suspension ◽

Internal Transcribed Spacer Region ◽

First Report ◽

Nitropropionic Acid ◽

First Case ◽

3 Nitropropionic Acid

Sugarcane (Saccharum officinarum L. cv. Badila) is a chewing cane cultivar in southern China. Since the first case of poisoning caused by the consumption of moldy sugarcane was confirmed in northern China in 1972, cases have occurred almost every year. It has been confirmed that Arthrinium is the pathogen that primarily occurs during improper postharvest storage (Liu xingjie, 1984). In 2019, ten moldy sugarcane stems (cv. Badila) were collected from Tang County, Baoding City, Hebei Province, China. The sugarcane flesh turned dark and was grayish-white, red, or reddish-brown. Some of them smelled musty. Symptomatic stems were surface disinfected using 75% ethanol and peeled aseptically. Small sections (3 mm3) were placed on potato dextrose agar amended with 0.01% chloramphenicol and incubated at 26 ± 2°C. Six fungal isolates were obtained from three sugarcane stems, a positive sample rate of 30%, and identified as the same fungus on the basis of morphological features owing to their formation of flat colonies that were initially white and later turned grayish white with moderate amounts of aerial mycelia. The mycelia consisted of smooth, hyaline, branched, and septate hyphae. The conidiophores were hyaline or pale brown and produced conidiogenous cells. The conidiogenous cells were pale brown, smooth, ampulliform, and 5.5 to 11.2 μm long (n=50). The conidia were brown, smooth, ellipsoidal to spherical, spherical in surface view, 4.5 to 7.4 μm in diameter, and 3.3 to 4.4 μm wide with a pale equatorial slit (n=50). The morphological characteristics of the one representative isolate, named LX1918, were identical to those of Arthrinium arundinis (Corda) Dyko & B. Sutton (Apiospora arundinis (Corda) Pintos & P. Alvarado) (Crous and Groenwald, 2013, Pintos and P. Alvarado, 2021). Genomic DNA was extracted from the mycelia to further identify the isolate. The internal transcribed spacer region (ITS rDNA), the translation elongation factor 1-alpha gene (TEF1) and the ß-tubulin gene (TUB2) were amplified using the primers ITS1/ITS4, EF1-728F/ EF-2 and T1/ Bt2b (White et al., 1990, O’Donnell et al. 1998, O’Donnell et al. 1997), respectively. BLASTn analysis of the ITS (556 bp, GenBank accession no. MW534386), TEF (434 bp, MW584370) and TUB2 (775 bp, MZ090019) sequences of isolate LX1918 showed that they were 99.43%, 99.52% and 99.74% similar to the published sequences of isolate CBS 106.12 (KF144883, KF145015 and KF144973), respectively. To confirm Koch’s postulates, pathogenicity tests were conducted in triplicate by inoculating the aseptic wounds with a conidial suspension (105/ml) of the isolate in healthy sugarcane stems. The controls were inoculated with sterile water. The sugarcane stems were incubated at 26 ± 2 °C and 86 % relative humidity in the dark. Obvious moldy symptoms appeared several days after the sugarcane stems had been inoculated. The sugarcane flesh turned reddish brown. In contrast, the control stems were asymptomatic. Ap. arundinis (Ar. arundinis) was reisolated from the inoculated and moldy sugarcane. In addition, 3-nitropropionic acid could be detected using HPLC-MS after the fungus had been cultured on potato yeast sucrose agar for 14 days. Previous studies had confirmed that 3-nitropropionic acid produced by Ar. sacchari, Ar. saccharicola and Ar. phaeospermum is the causal agent of poisoning caused by the consumption of moldy sugarcane (Hu wenjuan, 1986, Liu xingjie,1987). To our knowledge, this is the first report of Ap. arundinis (Ar. arundinis) as the causal agent of infected sugarcane and its production of 3-nitropropionic acid, which is toxic to humans. Therefore, the confirmation that Ap. arundinis(Ar. arundinis) infects sugarcane will expand our understanding of this pathogen and provide fundamental knowledge about the control of Apiospora mold to decrease the incidents of 3-nitropropionic acid poisoning.

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First Report of Zucchini Collapse by Fusarium solani f. sp. cucurbitae Race 1 and Plectosporium tabacinum in Italy

Plant Disease ◽

10.1094/pdis-91-3-0325a ◽

2007 ◽

Vol 91 (3) ◽

pp. 325-325 ◽

Cited By ~ 7

Author(s):

S. Vitale ◽

M. Maccaroni ◽

A. Belisario

Keyword(s):

Fusarium Solani ◽

Fusarium Species ◽

Morphological Characteristics ◽

Its Region ◽

Conidial Suspension ◽

Single Spore ◽

Running Water ◽

First Report ◽

Race 1 ◽

Rot Disease

Zucchini plant collapse has been often associated with Fusarium solani f. sp. cucurbitae race 1, which is the causal agent of Fusarium crown and foot rot disease of cucurbits. In Italy, F. solani f. sp. cucurbitae race 1 has been reported on zucchini (Cucurbita pepo) in a greenhouse in the Tuscany Region (4). In spring 2005, a severe outbreak was observed on zucchini in a vast area of cultivation in the province of Venice. Isolations from necrotic vessels gave more than 20 single-spore cultures. On the basis of morphological characteristics, they were identified as F. solani (2) and Plectosporium tabacinum (3). The internal transcribed spacer (ITS) region of rDNA was amplified and sequenced. A fragment of 454 and 531 bp was 99% homologous with sequence PSU66732 and AF150472 of F. solani f. sp. cucurbitae race 1 and P. tabacinum, respectively, in the NCBI database. The nucleotide sequences have been assigned Accession No. AM408782 for F. solani f. sp. cucurbitae race 1 and AM408781 for P. tabacinum. Pathogenicity tests were conducted with four isolates of each species on 15-day-old zucchini plants and on fruit. Plants were inoculated by dipping the roots in a conidial suspension of 106 spores ml-1 for 10 min. Control plants were dipped in sterile water. Five replicates for the inoculated and control plants were used. All plants were maintained in a greenhouse at approximately 24°C. After 14 days, inoculations with F. solani f. sp. cucurbitae race 1 gave symptoms of a cortical rot at the base of the stem with a progressive yellows and wilting of leaves, while plants inoculated with P. tabacinum displayed a moderate wilting. Fruit were washed under running water, disinfected with a solution of 3% sodium hypochlorite and 5% ethanol for 1 min, and inoculated with 6-mm-diameter mycelial plugs cut from the margin of 10-day-old cultures grown on PDA. Plugs were inserted into holes (approximately 2 mm deep) made with a sterile 7-mm-diameter cork borer. Five replicates per isolate were used. Fruit were kept at room temperature (22 to 24°C) in a moist chamber. All isolates induced symptoms of fruit rotting 10 days after inoculation. All controls remained healthy. The colonies reisolated from the inoculated plants and fruit were morphologically identical to the original isolates. The results obtained proved that F. solani f. sp. cucurbitae race 1 can be considered the major pathogen in zucchini collapse, at the same time P. tabacinum may play a role in this syndrome as reported for other cucurbits (1). To our knowledge, this is the first report of zucchini plant collapse caused by F. solani f. sp. cucurbitae race 1 and P. tabacinum, and the first report of P. tabacinum on zucchini in Italy. References: (1) V. J. Garcia-Jimenez et al. EPPO Bull. 30:169, 2000. (2) P. E. Nelson et al. Fusarium Species: An Illustrated Manual for Identification. Pennsylvania State University, University Park, 1983. (3) M. E. Palm et al. Mycologia 87:397, 1995. (4) G. Vannacci and P. Gambogi. Phytopathol. Mediterr. 19:103, 1980.

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First Report of Diaporthe longicolla Causing Leaf Spot on Kalanchoe pinnata in China

Plant Disease ◽

10.1094/pdis-12-20-2681-pdn ◽

2021 ◽

Author(s):

Xiaodong Sun ◽

Xinglai Cai ◽

Qiangqiang Pang ◽

Man Zhou ◽

Wen Zhang ◽

...

Keyword(s):

Leaf Spot ◽

Southern China ◽

Elongation Factor ◽

Morphological Characteristics ◽

Negative Control ◽

Hainan Province ◽

Conidial Suspension ◽

Single Spore ◽

First Report ◽

Kalanchoe Pinnata

Kalanchoe pinnata (Lam.) Pers. [syn.: Bryophyllum pinnatum (Lam.) Oken] is an important medicinal agent in southern China. The succulent leaves of this plant are used in the treatment of cholera, bruises, urinary diseases and whitlow. In Oct. 2019, leaf spots were detected on K. pinnata plants in Chengmai County, Hainan Province, China. Lesions with brown to black margins were irregularly shaped and associated with leaf margins. Spots coalesced to form larger lesions (Fig. S1-A), with black pycnidia present in more mature lesions. Symptomatic K. pinnata were found with 10-20% incidence during the humid winters of Hainan Province. Leaf tissues of 10 symptomatic plants were collected and surface sterilized in 70% ETOH for 30s, 0.1% HgCl2 for 30 s, rinsed 3x with sterile distilled water for 30s, placed on potato dextrose agar (PDA) amended with 30mg/L of kanamycin sulfate, and incubated at 25°C in the dark for 3-5 days. Four fungal isolates were obtained using a single-spore isolation method. The colonies were floccose, dense, and white with forming on older colonies grown on PDA (Fig. S1-B-1&2). Alpha conidia exuded from ostiole, rostrate, long-beaked pycnidia in creamy-to-yellowish drops. Alpha conidia were hyaline, ellipsoidal, separated and averaged 6.3μm (SD ± 1.13) long × 1.9μm (SD ± 0.33) wide (n=50). Beta conidia were not seen. The morphological characteristics matched the previous description of Diaporthe longicolla (syn. Phomopsis longicolla) (Hobbs et al. 1985). Mycelial genomic DNA of the representative isolate LDSG3-2 was extracted as template. The internal transcribed spacer (ITS) , translation elongation factor 1α gene (TEF) and β-tubulin (TUB2) regions were amplified. These loci were amplified using primer pairs ITS4/ITS5 (White, et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999) and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. A BLAST search of GenBank showed ITS (MN960195), TEF (MN974483) and TUB2 (MN974482) sequences of the isolate were 99%, 100%, and 99% homologous with D. longicolla strains DL11 (MF125048, 557/563 bp), D55 (MN584792, 347/347 bp) and DPC-HOH-32 (MK161506, 502/504 bp). Maximum likelihood trees based on concatenated nucleotide sequences of the three genes were constructed using MEGA 7.0, and bootstrap values indicated the isolate was D. longicolla (Fig. S1-D). Pathogenicity testing was performed using isolate LDSG3-2 by depositing 5µl droplets of a conidial suspension (1 × 106 ml-1) into 5 artificially wounded leaves (using a sterile needle) of 10 healthy 3-month-old K. pinnata plants. An equal number of artificially wounded control leaves were inoculated with sterile water to serve as a negative control. The test was conducted three times. Plants were kept at 25°C in 80% relative humidity and observed for symptoms. Two weeks after inoculation, no symptoms were observed on control plants (Fig. S1-C-1) and all inoculated plants showed symptoms (Fig. S1-C-2) similar to those observed in the field. The fungus was re-isolated from the infected tissues and showed the same cultural and morphological characteristics of the strain inoculated and could not be isolated from the controls fulfilling Koch’s postulates. To our knowledge, this is the first report of leaf spot on K. pinnata caused by D. longicolla in China. This disease is of concern since Phomopsis diseases are common in K. pinnata fields and can cause significant reduction in yield. References: White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA. DOI: 10.1016/0167-7799(90)90215-J Carbone, I., and Kohn, L. M. 1999. Mycologia. 91:553. DOI: 10.2307/3761358 Glass, N. L., and Donaldson, G. C. 1995. Appl. Environ. Microbiol. 61:1323. DOI: 10.1002/bit.260460112 Hobbs, T. W. et al. 1985. Mycologia. 77: 535. DOI: 10.2307/3793352

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First Report of Pepper Fruit Rot Caused by Fusarium concentricum in China

Plant Disease ◽

10.1094/pdis-03-13-0325-pdn ◽

2013 ◽

Vol 97 (12) ◽

pp. 1657-1657 ◽

Cited By ~ 6

Author(s):

J. H. Wang ◽

Z. H. Feng ◽

Z. Han ◽

S. Q. Song ◽

S. H. Lin ◽

...

Keyword(s):

Fusarium Species ◽

Fruit Rot ◽

Post Inoculation ◽

Distilled Water ◽

Conidial Suspension ◽

Average Growth ◽

First Report ◽

Control Fruit ◽

Pepper Fruit ◽

Fruit Samples

Pepper (Capsicum annuum L.) is an important vegetable crop worldwide. Some Fusarium species can cause pepper fruit rot, leading to significant yield losses of pepper production and, for some Fusarium species, potential risk of mycotoxin contamination. A total of 106 diseased pepper fruit samples were collected from various pepper cultivars from seven provinces (Gansu, Hainan, Heilongjiang, Hunan, Shandong, Shanghai, and Zhejiang) in China during the 2012 growing season, where pepper production occurs on approximately 25,000 ha. Pepper fruit rot symptom incidence ranged from 5 to 20% in individual fields. Symptomatic fruit tissue was surface-sterilized in 0.1% HgCl2 for 1 min, dipped in 70% ethanol for 30 s, then rinsed in sterilized distilled water three times, dried, and plated in 90 mm diameter petri dishes containing potato dextrose agar (PDA). After incubation for 5 days at 28°C in the dark, putative Fusarium colonies were purified by single-sporing. Forty-three Fusarium strains were isolated and identified to species as described previously (1,2). Morphological characteristics of one strain were identical to those of F. concentricum. Aerial mycelium was reddish-white with an average growth rate of 4.2 to 4.3 mm/day at 25°C in the dark on PDA. Pigments in the agar were formed in alternating red and orange concentric rings. Microconidia were 0- to 1-septate, mostly 0-septate, and oval, obovoid to allantoid. Macroconidia were relatively slender with no significant curvature, 3- to 5-septate, with a beaked apical cell and a foot-shaped basal cell. To confirm the species identity, the partial TEF gene sequence (646 bp) was amplified and sequenced (GenBank Accession No. KC816735). A BLASTn search with TEF gene sequences in NCBI and the Fusarium ID databases revealed 99.7 and 100% sequence identity, respectively, to known TEF sequences of F. concentricum. Thus, both morphological and molecular criteria supported identification of the strain as F. concentricum. This strain was deposited as Accession MUCL 54697 (http://bccm.belspo.be/about/mucl.php). Pathogenicity of the strain was confirmed by inoculating 10 wounded, mature pepper fruits that had been harvested 70 days after planting the cultivar Zhongjiao-5 with a conidial suspension (1 × 106 spores/ml), as described previously (3). A control treatment consisted of inoculating 10 pepper fruits of the same cultivar with sterilized distilled water. The fruit were incubated at 25°C in a moist chamber, and the experiment was repeated independently in triplicate. Initially, green to dark brown lesions were observed on the outer surface of inoculated fruit. Typical soft-rot symptoms and lesions were observed on the inner wall when the fruit were cut open 10 days post-inoculation. Some infected seeds in the fruits were grayish-black and covered by mycelium, similar to the original fruit symptoms observed at the sampling sites. The control fruit remained healthy after 10 days of incubation. The same fungus was isolated from the inoculated infected fruit using the method described above, but no fungal growth was observed from the control fruit. To our knowledge, this is the first report of F. concentricum causing a pepper fruit rot. References: (1) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA, 2006. (2) K. O'Donnell et al. Proc. Nat. Acad. Sci. USA 95:2044, 1998. (3) Y. Yang et al. 2011. Int. J. Food Microbiol. 151:150, 2011.

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First Report of Diaporthe hongkongensis Causing Fruit rot on Peach (Prunus persica) in China

Plant Disease ◽

10.1094/pdis-07-20-1505-pdn ◽

2021 ◽

Author(s):

Zhou Zhang ◽

Zheng Bing Zhang ◽

Yuan Tai Huang ◽

FeiXiang Wang ◽

Wei Hua Hu ◽

...

Keyword(s):

Prunus Persica ◽

Fruit Rot ◽

Hunan Province ◽

Post Inoculation ◽

Distilled Water ◽

Conidial Suspension ◽

Internal Transcribed Spacer Region ◽

First Report ◽

Representative Isolate ◽

Rot Disease

Peach [Prunus persica (L.) Batsch] is an important deciduous fruit tree in the family Rosaceae and is a widely grown fruit in China (Verde et al., 2013). In July and August 2018, a fruit rot disease was observed in a few peach orchards in Zhuzhou city, the Hunan Province of China. Approximately 30% of the fruit in more than 400 trees was affected. Symptoms displayed were brown necrotic spots that expanded, coalesced, and lead to fruit being rotten. Symptomatic tissues excised from the margins of lesions were surface sterilized in 70% ethanol for 10 s, 0.1% HgCl2 for 2 min, rinsed with sterile distilled water three times, and incubated on potato dextrose agar (PDA) at 26°C in the dark. Fungal colonies with similar morphology developed, and eight fungal colonies were isolated for further identification. Colonies grown on PDA were grayish-white with white aerial mycelium. After an incubation period of approximately 3 weeks, pycnidia developed and produced α-conidia and β-conidia. The α-conidia were one-celled, hyaline, fusiform, and ranged in size from 6.0 to 8.4 × 2.1 to 3.1 μm, whereas the β-conidia were filiform, hamate, and 15.0 to 27.0 × 0.8 to 1.6 μm. For molecular identification, total genomic DNA was extracted from the mycelium of a representative isolate HT-1 and the internal transcribed spacer region (ITS), β-tubulin gene (TUB), translation elongation factor 1-α gene (TEF1), calmodulin (CAL), and histone H3 gene (HIS) were amplified and sequenced (Meng et al. 2018). The ITS, TUB, TEF1, CAL and HIS sequences (GenBank accession nos. MT740484, MT749776, MT749778, MT749777, and MT749779, respectively) were obtained and in analysis by BLAST against sequences in NCBI GenBank, showed 99.37 to 100% identity with D. hongkongensis or D. lithocarpus (the synonym of D. hongkongensis) (Gao et al., 2016) (GenBank accession nos. MG832540.1 for ITS, LT601561.1 for TUB, KJ490551.1 for HIS, KY433566.1 for TEF1, and MK442962.1 for CAL). Pathogenicity tests were performed on peach fruits by inoculation of mycelial plugs and conidial suspensions. In one set, 0.5 mm diameter mycelial discs, which were obtained from an actively growing representative isolate of the fungus on PDA, were placed individually on the surface of each fruit. Sterile agar plugs were used as controls. In another set, each of the fruits was inoculated by application of 1 ml conidial suspension (105 conidia/ml) by a spray bottle. Control assays were carried out with sterile distilled water. All treatments were maintained in humid chambers at 26°C with a 12-h photoperiod. The inoculation tests were conducted twice, with each one having three fruits as replications. Six days post-inoculation, symptoms of fruit rot were observed on inoculated fruits, whereas no symptoms developed on fruits treated with agar plugs and sterile water. The fungus was re-isolated and identified to be D. hongkongensis by morphological and molecular methods, thus fulfilling Koch’s Postulates. This fungus has been reported to cause fruit rot on kiwifruit (Li et al. 2016) and is also known to cause peach tree dieback in China (Dissanayake et al. 2017). However, to our knowledge, this is the first report of D. hongkongensis causing peach fruit rot disease in China. The identification of the pathogen will provide important information for growers to manage this disease.

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First Report of Ramularia coleosporii causing Leaf Spot on Perilla frutescens in Korea

Plant Disease ◽

10.1094/pdis-01-21-0223-pdn ◽

2021 ◽

Author(s):

Md Aktaruzzaman ◽

Tania Afroz ◽

Hyo-Won Choi ◽

Byung Sup Kim

Keyword(s):

Leaf Spot ◽

Ipomoea Batatas ◽

Rust Fungus ◽

Morphological Characteristics ◽

Perilla Frutescens ◽

Herbaceous Plant ◽

Pathogenicity Test ◽

Conidial Suspension ◽

Single Spore ◽

First Report

Perilla (Perilla frutescens var. japonica), a member of the family Labiatae, is an annual herbaceous plant native to Asia. Its fresh leaves are directly consumed and its seeds are used for cooking oil. In July 2018, leaf spots symptoms were observed in an experimental field at Gangneung-Wonju National University, Gangneung, Gangwon province, Korea. Approximately 30% of the perilla plants growing in an area of about 0.1 ha were affected. Small, circular to oval, necrotic spots with yellow borders were scattered across upper leaves. Masses of white spores were observed on the leaf underside. Ten small pieces of tissue were removed from the lesion margins of the lesions, surface disinfected with NaOCl (1% v/v) for 30 s, and then rinsed three times with distilled water for 60 s. The tissue pieces were then placed on potato dextrose agar (PDA) and incubated at 25°C for 7 days. Five single spore isolates were obtained and cultured on PDA. The fungus was slow-growing and produced 30-50 mm diameter, whitish colonies on PDA when incubated at 25ºC for 15 days. Conidia (n= 50) ranged from 5.5 to 21.3 × 3.5 to 5.8 μm, were catenate, in simple or branched chains, ellipsoid-ovoid, fusiform, and old conidia sometimes had 1 to 3 conspicuous hila. Conidiophores (n= 10) were 21.3 to 125.8 × 1.3 to 3.6 μm in size, unbranched, straight or flexuous, and hyaline. The morphological characteristics of five isolates were similar. Morphological characteristics were consistent with those described for Ramularia coleosporii (Braun, 1998). Two representative isolates (PLS 001 & PLS003) were deposited in the Korean Agricultural Culture Collection (KACC48670 & KACC 48671). For molecular identification, a multi-locus sequence analysis was conducted. The internal transcribed spacer (ITS) regions of the rDNA, partial actin (ACT) gene and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene were amplified using primer sets ITS1/4, ACT-512F/ACT-783R and gpd1/gpd2, respectively (Videira et al. 2016). Sequences obtained from each of the three loci for isolate PLS001 and PLS003 were deposited in GenBank with accession numbers MH974744, MW470869 (ITS); MW470867, MW470870 (ACT); and MW470868, MW470871 (GAPDH), respectively. Sequences for all three genes exhibited 100% identity with R. coleosporii, GenBank accession nos. GU214692 (ITS), KX287643 (ACT), and 288200 (GAPDH) for both isolates. A multi-locus phylogenetic tree, constructed by the neighbor-joining method with closely related reference sequences downloaded from the GenBank database and these two isolates demonstrated alignment with R. coleosporii. To confirm pathogenicity, 150 mL of a conidial suspension (2 × 105 spores per mL) was sprayed on five, 45 days old perilla plants. An additional five plants, to serve as controls, were sprayed with sterile water. All plants were placed in a humidity chamber (>90% relative humidity) at 25°C for 48 h after inoculation and then placed in a greenhouse at 22/28°C (night/day). After 15 days leaf spot symptoms, similar to the original symptoms, developed on the leaves of the inoculated plants, whereas the control plants remained symptomless. The pathogenicity test was repeated twice with similar results. A fungus was re-isolated from the leaf lesions on the inoculated plants which exhibited the same morphological characteristics as the original isolates, fulfilling Koch’s postulates. R. coleosporii has been reported as a hyperparasite on the rust fungus Coleosporium plumeriae in India & Thailand and also as a pathogen infecting leaves of Campanula rapunculoides in Armenia, Clematis gouriana in Taiwan, Ipomoea batatas in Puerto Rico, and Perilla frutescens var. acuta in China (Baiswar et al. 2015; Farr and Rossman 2021). To the best of our knowledge, this is the first report of R. coleosporii causing leaf spot on P. frutescens var. japonica in Korea. This disease poses a threat to production and management strategies to minimize leaf spot should be developed.

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