First Report of Leptosphaeria biglobosa ‘brassicae’ Causing Blackleg on Brassica juncea var. multisecta in China

Brassica juncea var. multisecta, a leafy mustard, is widely grown in China as a vegetable (Fahey 2016). In May 2018, blackleg symptoms, grayish lesions with black pycnidia, were found on stems and leaves of B. juncea var. multisecta during disease surveys in Wuhan, Hubei Province. Disease incidence was approximately 82% of plants in the surveyed fields (~1 ha in total). To determine the causal agent of the disease, twelve diseased petioles were surface-sterilized and then cultured on potato dextrose agar (PDA) at 20˚C for 5 days. Six fungal isolates (50%) were obtained. All showed fluffy white aerial mycelia on the colony surface and produced a yellow pigment in PDA. In addition, pink conidial ooze formed on top of pycnidia after 20 days of cultivation on a V8 juice agar. Pycnidia were black-brown and globose with average size of 145 × 138 μm and ranged between 78 to 240 × 71 to 220 μm, n = 50. The conidia were cylindrical, hyaline, and 5.0 × 2.1 μm (4 to 7.1 × 1.4 to 2.9 μm, n=100). These results indicated that the fungus was Leptosphaeria biglobosa rather than L. maculans, as only the former produces yellow pigment (Williams and Fitt 1999). For molecular confirmation of identify, genomic DNAs were extracted and tested through polymerase chain reaction (PCR) assay using the species-specific primers LbigF, LmacF, and LmacR (Liu et al. 2006), of which DNA samples of L. maculans isolate UK-1 (kindly provided by Dr. Yongju Huang of University of Hertfordshire) and L. biglobosa ‘brassicae’ isolate B2003 (Cai et al. 2014) served as controls. Moreover, the sequences coding for actin, β-tubulin, and the internal transcribed spacer (ITS) region of ribosomal DNA (Vincenot et al. 2008) of isolates HYJ-1, HYJ-2 and HYJ-3 were also cloned and sequenced. All six isolates only produced a 444-bp DNA fragment, the same as isolate B2003, indicating they belonged to L. biglobosa ‘brassicae’, as L. maculans generates a 331-bp DNA fragment. In addition, sequences of ITS (GenBank accession no. MN814012, MN814013, MN814014), actin (MN814292, MN814293, MN814294), and β-tubulin (MN814295, MN814296, MN814297) of isolates HYJ-1, HYJ-2 and HYJ-3 were 100% identical to the ITS (KC880981), actin (AY748949), and β-tubulin (AY748995) of L. biglobosa ‘brassicae’ strains in GenBank, respectively. To determine their pathogenicity, needle-wounded cotyledons (14 days) of B. juncea var. multisecta ‘K618’ were inoculated with a conidial suspension (1 × 107 conidia/ml, 10 μl per site) of two isolates HYJ-1 and HYJ-3, twelve seedlings per isolate (24 cotyledons), while the control group was only treated with sterile water. All seedlings were incubated in a growth chamber (20°C, 100% relative humidity under 12 h of light/12 h of dark) for 10 days. Seedlings inoculated with conidia showed necrotic lesions, whereas control group remained asymptomatic. Two fungal isolates showing the same culture morphology to the original isolates were re-isolated from the necrotic lesions. Therefore, L. biglobosa ‘brassicae’ was confirmed to be the causal agent of blackleg on B. juncea var. multisecta in China. L. biglobosa ‘brassicae’ has been reported on many Brassica crops in China, such as B. napus (Fitt et al. 2006), B. oleracea (Zhou et al. 2019), B. juncea var. multiceps (Zhou et al. 2019), B. juncea var. tumida (Deng et al. 2020). To our knowledge this is the first report of L. biglobosa ‘brassicae’ causing blackleg on B. juncea var. multisecta in China, and its occurrence might be a new threat to leafy mustard production of China.

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First Report of Leptosphaeria biglobosa Causing Black Leg on Raphanus sativus in Central China

Plant Disease ◽

10.1094/pdis-07-13-0728-pdn ◽

2014 ◽

Vol 98 (7) ◽

pp. 993-993 ◽

Cited By ~ 4

Author(s):

X. Cai ◽

L. Yang ◽

J. Zhang ◽

G. Q. Li

Keyword(s):

Raphanus Sativus ◽

Disease Incidence ◽

Central China ◽

Conidial Suspension ◽

Pcr Assay ◽

First Report ◽

Dna Fragment ◽

Plant Pathol ◽

Black Leg ◽

Β Tubulin

Chinese radish (Raphanus sativus) is an important vegetable grown widely in China. In 2010 to 2013, surveys for Leptosphaeria spp. on cruciferous vegetables were conducted in 17 counties in Hubei Province, China. Black leg symptoms on tuber roots and basal stems of radish were observed in Hanchuan, Jingmen, and Macheng counties. Disease incidence ranged from 2 to 25% in 10 surveyed radish fields. Five fungal isolates were obtained from diseased radish plants by surface-sterilizing radish tissue (5% NaOCl for 90 s, and then rinsed in sterilized water three times) and plating onto potato dextrose agar (PDA) plates incubated at 20°C. The isolations produced fluffy white colonies with a yellow pigment, and black-brown, globose pycnidia with pink conidial ooze formed after 10 days. Pycnidia were 150 to 200 × 80 to 100 μm. Conidia were hyaline, cylindrical, and 4 to 5 × 2 μm. The cultural and morphological characteristics of the isolates matched the description for Phoma lingam, anamorph of Leptosphaeria maculans and L. biglobosa (3). All five isolates were identified by PCR assay using the species-specific primers LbigF, LmacF, and LmacR (1), and isolate HCLB-1 was identified further by PCR cloning and analysis of the sequences coding for actin, β-tubulin, and the internal transcribed spacer (ITS) region of ribosomal DNA (3,4). Genomic DNA was extracted from mycelium of each isolate harvested from 7-day-old cultures in V8 broth using the CTAB method (5). A 444-bp DNA fragment was detected by PCR assay, suggesting that all five isolates belonged to L. biglobosa rather than L. maculans as the latter generates a 331-bp DNA fragment (1). The HCLB-1 sequences for ITS (587 bp, GenBank Accession. No. KC880981), actin (899 bp, KF307762), and β-tubulin (432 bp, KF220296) genes were 99 to 100% identical to those of L. biglobosa isolates in GenBank. All five isolates were tested for pathogenicity on R. sativus cultivars Duan Ye 13 and Qi Ye Hong. Cotyledons of 10-day-old radish seedlings and post-harvest mature roots were wounded using a sterilized needle, and 10 μl of a conidial suspension (1 × 107 conidia/ml) of each isolate was pipetted onto the wounded area on each cotyledon or root, respectively, with 12 cotyledons (= 24 wounded sites) and 1 root (= 6 wounded sites) inoculated/isolate. One wounded root and 12 wounded cotyledons inoculated with water were used as control treatments. Treated roots and seedlings were incubated at 20°C and 100% RH in the dark for 7 days, and under a 12 h light/12 h dark cycle for 12 days, respectively. While the control roots and cotyledons remained asymptomatic, the roots and cotyledons inoculated with all five test isolates formed black cankers and necrotic lesions, respectively, in the inoculated wounds. A fungus re-isolated from symptomatic roots and cotyledons resembled the original isolates in colony morphology and the 444-bp DNA fragment detected by PCR assay. No fungus was isolated from control seedlings or roots. Thus, L. biglobosa appears to be the causal agent of black leg observed on radish in Hubei, China. L. biglobosa was reported to infect wild radish (R. raphanistrum) (2). To our knowledge, this is the first report of L. biglobosa causing black leg on R. sativus. References: (1) S. Y. Liu et al. Plant Pathol. 55:401, 2006. (2) A. Maxwell and J. K. Scott. Australas. Plant Pathol. 37:523, 2008. (3) L. Vincenot et al. Phytopathology 98:321, 2008. (4) T. J. White et al. PCR Protocols: A Guide to Methods and Applications, Academic Press, 1990. (5) J. Zhang et al. Mycologia 102:1114, 2010.

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First Report of Colletotrichum cereale Causing Anthracnose on Avena nuda in China

Plant Disease ◽

10.1094/pdis-10-20-2109-pdn ◽

2020 ◽

Author(s):

Na Zhao ◽

Junyu Yang ◽

Xiaoli Fang ◽

lingrui Li ◽

Hongfei Yan ◽

...

Keyword(s):

Morphological Characteristics ◽

Causal Agent ◽

Post Inoculation ◽

Pathogenicity Test ◽

Conidial Suspension ◽

Single Spore ◽

First Report ◽

Fungal Isolates ◽

Colletotrichum Cereale ◽

Avena Nuda

Naked oats (Avena nuda L.) is rich in protein, fat, vitamin, mineral elements and so on, and is one of the world's recognized cereal crops with the highest nutritional and healthcare value. In July 2019, leaf spot was detected on A. nuda in Zhangbei experimental station of Hebei Agricultural University. The incidence of disease is 10% to 20%. The symptoms were similar to anthracnose disease, the infected leaves had fusiform or nearly fusiform yellowish-brown spots, yellow halo around the spots. Numerous acervuli with black setae diagnostic of fungi in the genus Colletotrichum were present on necrotic lesions. To identify the pathogen, ten symptomatic leaves were collected, and only one disease spot was isolated from each leaf. Small square leaf pieces (3 to 5 mm) were excised from the junction of diseased and healthy tissues with a sterile scalpel and surface disinfested with 75% alcohol for 30s, 0.1% corrosive sublimate for 1 min, rinsed three times in sterile water. Plant tissues were then transferred on potato dextrose agar (PDA), and incubated at 25°C for 7 days. Two fungal isolates were obtained and purified by single-spore isolation method. All fungi have the same morphology and no other fungi were isolated. The aerial mycelium was gray black. The conidia were colorless and transparent, falcate, slightly curved, tapered toward the tips, and produced in acervuli with brown setae. The length and width of 100 conidia were measured and size ranged from 1.86 to 3.84 × 8.62 to 29.81 μm. These morphological characteristics were consistent with the description of Colletotrichum cereale (Crouch et al. 2006). To further assess the identity of the species, the genomic DNA of two fungal isolates (LYM19-4 and LYM19-10) was extracted by a CTAB protocol. The ribosomal DNA internal transcribed spacer (ITS) region as well as, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), and the beta-tubulin 2 (Tub2) partial genes were amplified and sequenced with primers ITS4/5, GDF/GDR, ACT-512F/ACT-783R, and T1/Bt2b, respectively (Carbone et al. 1999; Templeton et al. 1992; O'Donnell et al. 1997; Glass et al. 1995). The sequences of the ITS-rDNA region (MW040121, MW040122), the GAPDH sequences (MW052554, MW052555), the ACT sequences (MW052556, MW052551) and the Tub2 sequences (MW052552, MW052553) of the two single-spore isolates were more than 99% identical to C. cereale isolate CGMCC3.15110 (JX625159, KC843517, KC843534 and JX625186). Maximum likelihood tree based on concatenated sequences of the four genes were constructed using MEGA7. The results showed the strains isolated from A. nuda were closely related to C. cereale, as supported by high bootstrap values. A pathogenicity test of the C. cereale isolates was performed on first unfolding leaves of A. nuda. Koch's postulates were carried out with isolates by spraying a conidial suspension of 106 conidia/mL on leaves of healthy A. nuda. Four replicated pots were inoculated at a time, 10 leaves each pot, while sterile distilled water was used as the control. All treated plants were placed in a moist chamber (25°C, 16-h light and 8-h dark period). Anthracnose symptoms developed on the inoculated plants 7 days post inoculation while all control plants remained healthy. Microscopic examination showed the surface of infected leaves had the same acervuli, setae, and conidia as the original isolate. The pathogenicity test was repeated three times. C. cereale was previously reported as the causal agent of anthracnose on feather reed grass in US (Crouch et al. 2009). To our knowledge, this is the first report of C. cereale as the causal agent of A. nuda anthracnose in China.

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First Report of Leptosphaeria biglobosa (Blackleg) on Brassica oleracea (Cabbage) in Mexico

Plant Disease ◽

10.1094/pdis-94-6-0791c ◽

2010 ◽

Vol 94 (6) ◽

pp. 791-791 ◽

Cited By ~ 10

Author(s):

A. Dilmaghani ◽

M. H. Balesdent ◽

T. Rouxel ◽

O. Moreno-Rico

Keyword(s):

Brassica Oleracea ◽

Sequence Data ◽

Leptosphaeria Maculans ◽

The United States ◽

Internal Transcribed Spacers ◽

First Report ◽

5.8S Rdna ◽

Single Location ◽

Leptosphaeria Biglobosa ◽

Β Tubulin

Broccoli (Brassica oleracea var. italica), cauliflower (B. oleracea var. botrytis), and cabbage (B. oleracea var. capitata) have been grown in central Mexico since 1970, with 21,000 ha cropped in 2001. In contrast, areas grown with oilseed rape (B. napus) are very limited in Mexico (<8,000 ha). Blackleg, a destructive disease of B. napus in most parts of the world, was first observed in Mexico in Zacatecas and Aguascalientes in 1988 on B. oleracea, causing as much as 70% yield loss. A species complex of two closely related Dothideomycete species, Leptosphaeria maculans and L. biglobosa, is associated with this disease of crucifers (1), but leaf symptoms on susceptible plants are different, with L. maculans typically causing >15-mm pale gray lesions with numerous pycnidia, whereas L. biglobosa causes dark and smaller lesions only containing a few pycnidia. Having a similar epidemiology, both species can be present on the same plants at the same time, and symptom confusion can occur as a function of the physiological condition of the plant or expression of plant resistance responses. A total of 209 isolates from symptomatic B. oleracea leaves were collected from three fields in central states of Mexico (58 to 71 isolates per location). All leaves showed similar symptoms, including a 10- to 15-mm tissue collapse with an occasional dark margin. Cotyledons of seven B. napus differentials were inoculated with conidia of all the isolates as described by Dilmaghani et al. (1). Two hundred isolates caused tissue collapse typical of L. maculans. However, nine obtained from white cabbage in a single location in Aguascalientes caused <5-mm dark lesions. When inoculated onto cotyledons of three B. oleracea genotypes commonly grown in Mexico (cvs. Domador, Monaco, and Iron Man), the nine isolates caused a range of symptoms characterized by tissue collapse (maximum 10 to 15 mm), showing the presence of patches of black necrotic spots within the collapse. The occasional presence of a few pycnidia allowed us to reisolate the fungus for molecular identification. ITS1-5.8S-ITS2, (internal transcribed spacers and 5.8S rDNA), actin, and β-tubulin sequences were obtained as described previously (4). Multiple gene genealogies based on these sequence data showed two subclades of L. biglobosa: L. biglobosa ‘occiaustralensis’ (one isolate; ITS [AM410082], actin [AM410084], and β-tubulin [AM410083]) and L. biglobosa ‘canadensis’ (eight isolates; ITS [AJ550868], actin [AY748956], and β-tubulin [AY749004]) (3,4), which were previously described on B. napus in the United States, Canada, and Chile. To our knowledge, this is the first report of L. biglobosa in Mexico. Previously, this species has only been reported once on B. oleracea without discrimination into subclades (2). In the Aguascalientes sampling, 24% of the isolates were L. biglobosa, similar to Canadian locations where this species is still common as compared with L. maculans (1). The large proportion of sampled L. biglobosa ‘canadensis’, highlights the prevalence of this subclade throughout the American continent (1). References: (1) A. Dilmaghani et al. Plant Pathol. 58:1044, 2009. (2) E. Koch et al. Mol. Plant-Microbe Interact. 4:341, 1991. (3) E. Mendes-Pereira et al. Mycol Res. 107:1287, 2003. (4) L. Vincenot et al. Phytopathology 98:321, 2008.

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First report of Leptosphaeria biglobosa ‘canadensis’ causing blackleg on oilseed rape (Brassica napus) in China

Plant Disease ◽

10.1094/pdis-12-20-2735-pdn ◽

2021 ◽

Author(s):

Tao Luo ◽

Guoqing Li ◽

Long Yang

Keyword(s):

Brassica Napus ◽

Oilseed Rape ◽

Southern China ◽

Disease Incidence ◽

Morphological Characteristics ◽

Gansu Province ◽

Bootstrap Support ◽

First Report ◽

Fungal Isolates ◽

Leptosphaeria Biglobosa

Oilseed rape (Brassica napus L.) is one of the most important oilseed crops in China. It is widely cultivated in China, with winter oilseed rape in Yangtze River basin and in southern China, and spring oilseed rape in northern China. In August 2017, a survey for Leptosphaeria spp. on spring oilseed rape was conducted in Minle county, Zhangye city, Gansu Province, China. The symptoms typical of blackleg on basal stems of oilseed rape were observed in the field. A large number of black fruiting bodies (pycnidia) were present on the lesions (Fig. 1A). The disease incidence of basal stem infection in the surveyed field was 19%. A total of 19 diseased stems were collected to isolate the pathogen. After surface sterilizing (75% ethanol for 30 s, 5% NaOCl for 60 s, followed by rinsing in sterilized water three times), diseased tissues were cultured on acidified potato dextrose agar (PDA) plates at 20°C for 7 days. Twelve fungal isolates were obtained. All fungal isolates produced typical tan pigment on PDA medium, and produced pycnidia after two weeks (Fig. 1B). Colony morphological characteristics indicated that these isolates might belong to Leptosphaeria biglobosa. To confirm identification, multiple PCR was conducted using the species-specific primers LmacF, LbigF, LmacR (Liu et al. 2006). Genomic DNA of each isolate was extracted using the cetyltrimethylammonium bromide (CTAB) method. DNA samples of L. maculans isolate UK-1 and L. biglobosa isolate W10 (Cai et al. 2015) were used as references. Only a 444-bp DNA band was detected in all 12 isolates and W10, whereas a 333-bp DNA band was detected only in the UK-1 isolate (Fig. 1C). PCR results suggested that these 12 isolates all belong to L. biglobosa. In addition, the internal transcribed spacer (ITS) region of these 12 isolates was analyzed for subspecies identification (Vincenot et al. 2008). Phylogenetic analysis based on ITS sequence showed that five isolates (Lb1134, Lb1136, Lb1138, Lb1139 and Lb1143) belonged to L. biglobosa ‘brassicae’ (Lbb) with 78% bootstrap support, and the other seven isolates (Lb1135, Lb1137, Lb1140, Lb1141, Lb1142, Lb1144 and Lb1145) belonged to L. biglobosa ‘canadensis’ (Lbc) with 95% bootstrap support (Fig. 1D). Two Lbb isolates (Lb1134 and Lb1136) and two Lbc isolates (Lb1142 and Lb1144) were randomly selected for pathogenicity testing on B. napus cultivar Zhongshuang No. 9 (Wang et al. 2002). Conidial suspensions (10 μL, 1 × 107 conidia mL-1) of these four isolates were inoculated on needle-wounded cotyledons (14-day-old seedling), with 10 cotyledons (20 wounded sites) per isolate. A further 10 wounded cotyledons were inoculated with water and served as controls. Seedlings were maintained in a growth chamber at 20°C with 100% relative humidity and a 12-h photoperiod. After 7 days, cotyledons inoculated with the four isolates showed necrotic lesions in the inoculated wounds. Control cotyledons had no symptoms (Fig. 2). Fungi re-isolated from the infected cotyledons showed similar colony morphology as the original isolates. Therefore, L. biglobosa ‘brassicae’ and L. biglobosa ‘canadensis’ appear to be the pathogens causing the observed blackleg symptoms on spring oilseed rape in Gansu, China. In previous studies, L. biglobosa ‘brassicae’ has been found in many crops in China, including oilseed rape (Liu et al. 2014; Cai et al. 2015), Chinese radish (Raphanus sativus) (Cai et al. 2014a), B. campestris ssp. chinensis var. purpurea (Cai et al. 2014b), broccoli (B. oleracea var. italica) (Luo et al. 2018), ornamental kale (B. oleracea var. acephala) (Zhou et al. 2019a), B. juncea var. multiceps (Zhou et al. 2019b), B. juncea var. tumida (Deng et al. 2020) and Chinese cabbage (B. rapa subsp. pekinensis) (Yu et al. 2021 accepted). To the best of our knowledge, this is the first report of L. biglobosa ‘canadensis’ causing blackleg on B. napus in China.

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First Report of Colletotrichum boninense Causing Anthracnose on Pepper in China

Plant Disease ◽

10.1094/pdis-04-12-0403-pdn ◽

2013 ◽

Vol 97 (1) ◽

pp. 138-138 ◽

Cited By ~ 7

Author(s):

Y. Z. Diao ◽

J. R. Fan ◽

Z. W. Wang ◽

X. L. Liu

Keyword(s):

Internal Transcribed Spacer ◽

Severe Disease ◽

Causal Agent ◽

Its Sequences ◽

Universal Primers ◽

Pathogenicity Test ◽

Conidial Suspension ◽

Single Spore ◽

First Report ◽

Species Specific

Anthracnose, caused by Colletotrichum spp., is a severe disease and results in large losses in pepper (Capsicum frutescens) production in China (4). Colletotrichum boninense is one of the Colletotrichum species in pepper in China. In August 2011, anthracnose symptoms (circular, sunken lesions with orange to black spore masses) were observed on pepper fruits in De-Yang, Sichuan Province, China. Three single-spore isolates (SC-6-1, SC-6-2, SC-6-3) were obtained from the infected fruits. A 5-mm diameter plug was transferred to potato dextrose agar (PDA); the isolates formed colonies with white margins and circular, dull orange centers. The conidia were cylindrical, obtuse at both ends, and 10.5 to 12.6 × 4.1 to 5.0 μm. The colonies grew rapidly at 25 to 28°C, and the average colony diameter was 51 to 52 mm after 5 days on PDA at 25°C. Based upon these characters, the causal agent was identified as C. boninense. To confirm the identity of the isolates, the internal transcribed spacer (ITS) regions were amplified with the ITS1/ITS4 universal primers (1). The internal transcribed spacer (ITS) sequences (Accession No. JQ926743) of the causal fungus shared 99 to 100% homology with ITS sequences of C. boninense in GenBank (Accession Nos. FN566865 and EU822801). The identity of the causal agent as C. boninense was also confirmed by species-specific primers (Col1/ITS4) (2). In a pathogenicity test, five detached ripe pepper fruits were inoculated with 1 μl of a conidial suspension (106 conidia/mL) or five fruits with 1 μl of sterile water were kept as control. After 7 days in a moist chamber at 25°C, typical anthracnose symptoms had developed on the five inoculated fruits but not on control fruits. C. boninense was reisolated from the lesions, and which was confirmed by morphology and molecular methods as before. There have reports of C. boninense infecting many species of plants, including pepper (3). To our knowledge, this is the first report of C. boninense causing anthracnose on pepper in China. References: (1) A. K. Lucia et al. Phytopathology 93:581, 2002. (2) S. A. Pileggi et al. Can. J. Microbiol. 55:1081, 2009. (3) H. J. Tozze et al. Plant Dis. 93:106, 2009. (4) M. L. Zhang. J. Anhui Agri. Sci. 2:21, 2000.

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First Report of Stem Blight Caused by Calonectria colhounii (Anamorph Cylindrocladium colhounii) on Greenhouse-Grown Blueberries in the United States

Plant Disease ◽

10.1094/pdis-02-11-0117 ◽

2011 ◽

Vol 95 (9) ◽

pp. 1187-1187

Author(s):

J. J. Sadowsky ◽

T. D. Miles ◽

A. M. C. Schilder

Keyword(s):

United States ◽

North Carolina ◽

Root Rot ◽

The United States ◽

Vaccinium Corymbosum ◽

Conidial Suspension ◽

Centraalbureau Voor ◽

First Report ◽

Stem Lesions ◽

Β Tubulin

Necrotic stems and leaves were observed on 2- to 4-month-old, rooted microshoot plants (Vaccinium corymbosum L. ‘Liberty’ and ‘Bluecrop’, V. angustifolium Aiton ‘Putte’, and V. corymbosum × V. angustifolium ‘Polaris’) in a Michigan greenhouse in 2008 and 2009. As the disease progressed, leaves fell off and 80 to 100% of the plants died in some cases. Root rot symptoms were also observed. A fungus was isolated from stem lesions. On potato dextrose agar (PDA), cultures first appeared light tan to orange, then rusty brown and zonate with irregular margins. Chains of orange-brown chlamydospores were abundant in the medium. Macroconidiophores were penicillately branched and had a stipe extension of 220 to 275 × 2.5 μm with a narrowly clavate vesicle, 3 to 4 μm wide at the tip. Conidia were hyaline and cylindrical with rounded ends, (1-)3-septate, 48 to 73 × 5 to 7 (average 60 × 5.5) μm and were held together in parallel clusters. Perithecia were globose to subglobose, yellow, 290 to 320 μm high, and 255 to 295 μm in diameter. Ascospores were hyaline, 2- to 3-septate, guttulate, fusoid with rounded ends, slightly curved, and 30 to 88 × 5 to 7.5 (average 57 × 5.3) μm. On the basis of morphology, the fungus was identified as Calonectria colhounii Peerally (anamorph Cylindrocladium colhounii Peerally) (1,2). The internal transcribed spacer region (ITS1 and ITS2) of the ribosomal DNA and the β-tubulin gene were sequenced (GenBank Accession Nos. HQ909028 and JF826867, respectively) and compared with existing sequences using BLASTn. The ITS sequence shared 99% maximum identity with that of Ca. colhounii CBS 293.79 (GQ280565) from Java, Indonesia, and the β-tubulin sequence shared 97% maximum identity with that of Ca. colhounii CBS 114036 (DQ190560) isolated from leaf spots on Rhododendron sp. in North Carolina. The isolate was submitted to the Centraalbureau voor Schimmelcultures in the Netherlands (CBS 129628). To confirm pathogenicity, 5 ml of a conidial suspension (1 × 105/ml) were applied as a foliar spray or soil drench to four healthy ‘Bluecrop’ plants each in 10-cm plastic pots. Two water-sprayed and two water-drenched plants served as controls. Plants were misted intermittently for 2 days after inoculation. After 7 days at 25 ± 3°C, drench-inoculated plants developed necrotic, sporulating stem lesions at the soil line, while spray-inoculated plants showed reddish brown leaf and stem lesions. At 28 days, three drench-inoculated and one spray-inoculated plant had died, while others showed stem necrosis and wilting. No symptoms were observed on control plants. Fungal colonies reisolated from surface-disinfested symptomatic stem, leaf, and root segments appeared identical to the original isolate. Cy. colhounii was reported to cause a leaf spot on blueberry plants in nurseries in China (3), while Ca. crotalariae (Loos) D.K. Bell & Sobers (= Ca. ilicicola Boedijn & Reitsma) causes stem and root rot of blueberries in North Carolina (4). To our knowledge, this is the first report of Ca. colhounii causing a disease of blueberry in Michigan or the United States. Because of its destructive potential, this pathogen may pose a significant threat in blueberry nurseries. References: (1) P. W. Crous. Taxonomy and Pathology of Cylindrocladium (Calonectria) and Allied Genera. The American Phytopathological Society, St. Paul, MN, 2002. (2) L. Lombard et al. Stud. Mycol. 66:31, 2010. (3) Y. S. Luan et al. Plant Dis. 90:1553, 2006. (4) R. D. Milholland. Phytopathology 64:831, 1974.

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First Report of Fusarium solani on Utah Sweetvetch in the United States

Plant Disease ◽

10.1094/pdis-08-12-0789-pdn ◽

2013 ◽

Vol 97 (3) ◽

pp. 423-423 ◽

Cited By ~ 2

Author(s):

S. Uppala ◽

B. M. Wu ◽

T. N. Temple

Keyword(s):

Native American ◽

Fusarium Solani ◽

Vascular Tissue ◽

Agricultural Research ◽

Apical Cell ◽

The United States ◽

Conidial Suspension ◽

Soil Dna ◽

First Report ◽

Fungal Isolates

Utah sweetvetch (Hedysarum boreale Nutt.) is a native American perennial nitrogen fixing legume used mainly in rangeland reclamation, soil rejuvenation, and erosion control. In June 2011, a field of Utah sweetvetch grown for seeds in central Oregon had approximately 15% of the plants exhibiting chlorosis, defoliation, stunting, wilting, and/or death. Dissection of the crown of symptomatic plants revealed discolored pinkish brown vascular tissue. Symptomatic tissues from six random plants were surface sterilized, placed on acidified potato dextrose agar (PDA) medium, and cultured for 7 days at room temperature, which allowed six fungal isolates (SS1 through SS6) to be collected. On PDA, all six isolates had rapid, creamy white colored growth. Based on observations of 1-week-old isolates, microconidia were oval to kidney shaped, single celled, 8 to 10 × 2.5 to 4 μm, and formed at the tips of long unbranched monophialides. Macroconidia were three to four septate, cylindrical to slightly curved, with characteristic foot shaped basal cell and blunt apical cell, 37 to 49 × 4.4 to 5.3 μm. Chlaymydospores observed were 8.5 to 11 × 7.6 to 9 μm. Based on fungal references (1,2,3), the isolates were identified as Fusarium solani (Mart.) Sacc. Identification of the isolates at the molecular level was determined by amplification of the internal transcribed spacer (ITS) region using PCR and amplicon sequencing. Botrytis cinerea and F. graminearum cultures were used as controls for the extraction, amplification, and sequencing steps. Genomic DNA was extracted from mycelia using protocols of the MOBIO Ultraclean Soil DNA Isolation Kit (MO-BIO Laboratories Inc, Carlsbad, CA, USA). PCR was performed using ITS1/ITS4 primers and resulted in 563- to 573-bp amplicons, which were sequenced. Analysis of the ITS sequences (GenBank Accession Nos. JX524018 to JX524023) for the six fungal isolates using BLASTn revealed a 99% sequence identity with F. solani strains (AB470903, AB513851, AJ608989, EF152426, EU029589, and HM214456). Pathogenicity was confirmed on Utah sweetvetch plants in the greenhouse. Seeds of Utah sweetvetch were first plated on acidified PDA for germination; healthy seedlings were then selected and transplanted into pots with sterilized soil after 2 weeks of growth. The plants were kept in a greenhouse at Central Oregon Agricultural Research Center, Madras, Oregon. Ten 40-day-old healthy vetch plants were inoculated by drenching with a mixed conidial suspension (107 conidia/ml) of the six F. solani isolates. Ten plants drenched with sterile distilled water were included as controls. Symptoms of chlorosis and stunting similar to those in the commercial field were observed within 30 days of inoculation on 8 of 10 inoculated plants, while control plants were symptomless. Fungal isolates identical to F. solani were reisolated from the symptomatic plants. To our knowledge, this is the first report of F. solani on Utah sweetvetch plants. References: (1) C. Booth. The Genus Fusarium. CMI, Kew, Surrey, UK, 1971. (2) P. E. Nelson et al. Fusarium species: An illustrated manual for identification. The Pennsylvania State University Press, USA, 1983. (3) H. I. Nirenberg. A simplified method for identifying Fusarium spp. occurring on wheat. Can. J. Bot. 59:1599, 1980.

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First report of Fusarium avenaceum causing Leaf Spot on Angelica sinensis in China

Plant Disease ◽

10.1094/pdis-08-21-1753-pdn ◽

2021 ◽

Author(s):

Zikun Zhang ◽

Jianqiang Zhang ◽

Wanxia Zhang ◽

Zhian Kou ◽

Xinfang Wang ◽

...

Keyword(s):

Leaf Spot ◽

Chinese Herb ◽

Gansu Province ◽

Fusarium Avenaceum ◽

Angelica Sinensis ◽

Conidial Suspension ◽

Data Set ◽

First Report ◽

Initial Symptoms ◽

Β Tubulin

Angelica sinensis (Oliv) Diels (Umbelliferae) is a popular Chinese herb that is mainly distributed in Gansu Province, China, accounting for more than 90% of the national output and sales. A survey for diseases of A. sinensis in Gansu Province in August 2019 found foliar disease with an incidence of 60 to100%, and severities ranging from 5 to 15%. The disease mainly occurred in late July and August. The initial symptoms included many light brown, small lesions, round or irregular in shape, which gradually increased in size. White mycelia was visible in the lesions. Severely affected leaves became chlorotic, withered and died. In the Angelica planting area in Weiyuan County (33°26′N, 104°02′E) diseased leaves from 20 plants were collected by the five-point sampling method (Zheng et al. 2018), and small samples (4 × 4 mm2) wee cut from the border between diseased and healthy tissue, successively sterilized with 75% ethanol for 30 sec, washed three times with sterilized water and dried on sterilized filter paper, and placed on potato dextrose agar plates. After 5 days at 25°C, five morphologically similar colonies were obtained. Colonies were somewhat round with pink overall and formed abundant fluffy white mycelium in the center. Conidia were solitary, macrospores slender, straight to slightly falcate with 2 to 6 septa, and ranged from 20.0 to 77.6 µm × 2.5 to 3.6 µm (n=50). The microspores were elliptical and ranged from 3.0 to 8.0 µm × 2.5 to 3.0 µm (n=5). The strong pink pigment was observed on the reverse side of the PDA culture. The morphological characteristics were consistent with the description of Fusarium avenaceum (Parikh et al. 2018; Jahedi et al. 2019). To further identify the strains, the internal transcribed spacer (ITS), β-tubulin, translation elongation factor 1α (EF1-α), and RNA polymerase second largest subunit (RPB2) gene regions were amplified with ITS1/ITS4, Bt2a/Bt2b, EF1/EF2, and 5f2/7cr (Glass and Donaldson 1995; O’Donnell et al. 2010; White et al. 1990), respectively. The sequences of the five strains were identical, and that of representative strain K0721 were deposited in GenBank (ITS, MZ389899; TUB2, MZ398139; EF1-α, MZ388462; RPB2, MZ394004). BLAST analysis revealed that the ITS, β-tubulin, EF1-α, and RPB2 sequences were 100% (563/563), 100% (423/423), 99% (643/649), and 99% (930/935) homology, with those of F. avenaceum (KP295511.1, KY475586.1, KU999088.1, and MH582082.1), respectively. A multigene phylogenetic tree was inferred by Maximum likelihood phylogenetic analyses based on the combined data set with ITS, EF1-α and RPB2. The strain K0721 was clustered with F. avenaceum. Pathogenicity tests were performed on five 1-month-old healthy plants in plastic pots (20 cm. diam.) with sterilized soil. Each was sprayed with 50 μl of a conidial suspension (1×104 conidia/mL), and 5 healthy plants were sprayed with sterile water as controls. Small lesions were observed after 5 days at 25℃ in a greenhouse. Symptoms were similar to those observed under field conditions. Control plants remained symptomless. Six isolates were reisolated from infected leaves and all confirmed to be F. avenaceum based on morphological observations and molecular identification. To our knowledge, only Septoria anthrisci has been previously reported as a pathogen of A. sinensis leaf spot (Wang et al. 2018), and this is the first report of F. avenaceum causing this disease. This discovery needs to be considered in developing and implementing disease management programs in A. sinensis production.

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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 Postharvest Stem End Rot of mango fruit (Mangifera indica) caused by Lasiodiplodia theobromae in China.

Plant Disease ◽

10.1094/pdis-01-21-0168-pdn ◽

2021 ◽

Author(s):

Yanling Ma ◽

Tanvir Ahmad ◽

Yongquan Zheng ◽

Nie Chengrong ◽

Yang Liu

Keyword(s):

Mangifera Indica ◽

Causal Agent ◽

Post Inoculation ◽

Mango Fruit ◽

Isolation Technique ◽

Lasiodiplodia Theobromae ◽

First Report ◽

Fruit Samples ◽

Fruit Surface ◽

Β Tubulin

China is the second largest producer of mango in the world, a fruit has high nutritive value and a rich source of fiber (Kuhn et al., 2017). In late June 2019, a postharvest stem-end rot disease was observed in different local fruit markets (39°48'42.1"N 116°20'17.0"E) of the Fengtai district of Beijing, China. Black rot symptomatic lesions were observed on the fruit surface which initially started from the stem end of the mango fruit (Fig. 1). Approximately 45 % of mango fruits were affected with the disease. Symptomatic portions from collected fruit samples (n=40) were cut into small pieces (2mm2), rinsed with 1% NaClO for 20s and then washed three times with sterilized distilled water (SDW) for surface disinfection. The disinfected pieces were then placed on sterilized filter paper for drying. Later, these pieces were placed on Potato Dextrose Agar (PDA) plates and incubated at 28°C for seven days. The resulting fungal colonies were purified by the single spore isolation technique. The isolated fungal colonies were initially greenish to gray in color, later turning olive-black to black. Conidia were dark brown in color, oval-shaped, two-celled and measured 22.4 to 25.7 (24.06 ± 0.15) μm in length and 10.2 to 12.8 (11.3 ± 0.13) μm in width (n=36). Based on the symptoms, culture morphology and microscopic characters, Lasiodiplodia theobromae was suspected as the causal agent, and similar results were reported by Pavlic et al., 2004 and Burgess et al., 2006. For molecular identification, a multi-locus sequence analysis approach was used. The Internal Transcribed Spacers (ITS) region, elongation factor 1 alpha (EF1-α) and β-tubulin genes were amplified and sequenced using ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn, 1999), and Bt2a/Bt2b (Glass and Donaldson, 1995) primers respectively. The sequences of isolate MFT9 were deposited to GenBank (MW115977 (ITS), (MW118595 (EF1-α) and MW118596 (β-tubulin). All sequences showed more than 99.5% similarity with reported sequences of Lasiodiplodia theobromae isolate IBL340 with accessions numbers KT247466 (ITS), KT247472 (EF1-α) and KT247475 (β-tubulin). Phylogenetic reconstruction based on Maximum Likelihood, using Mega X (Kumar et al., 2018), grouped isolate MFT9 with isolates representing L. theobromae. Pathogenicity testing was performed on 18 fresh, healthy, medium-sized mango fruits for each treatment to fulfill Koch’s postulate. The fruits were disinfested with 1% NaClO and punctured with a sterilized needle to create approximately 2mm2 wounds for inoculation. Fruits were inoculated with 15µL of fresh inoculum (107 spores/mL) from isolate MFT9. Control fruits were inoculated with 15µL of SDW and both the inoculated and control fruits were incubated at 28°C for seven days of post inoculation. The rot lesions appeared at the point of inoculation and gradually spread on the fruit surface. The symptoms were similar to the symptoms observed on the original fruit samples (Fig. 2). This experiment was conducted three times under the same conditions, with control fruits remaining asymptomatic each time. The re-isolated fungus was identified as L. theobromae based on symptoms and morpho-molecular analysis, described above. L. theobromae is also reported as a causal agent responsible for a postharvest stem-end rot on Coconut in China (Zhang, et al., 2019). To our knowledge, this is the first report of L. theobromae causing postharvest stem-end rot of mango fruit in China. This finding suggests that L. theobromae is a potential problem for mango fruit production in China.

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