scholarly journals First Report of Southern Blight Caused by Sclerotium delphinii on Euonymus fortunei in China

Plant Disease ◽  
2021 ◽  
Author(s):  
Ronghua Sun ◽  
Guangliang Lu ◽  
Yuezhong Li ◽  
Qingquan Luo

Euonymus fortunei is an evergreen shrub-vine in the family Celastraceae, widely used as a groundcover or a vine to climb walls, or traditional herbal medicine in China. In August 2019, typical southern blight symptoms that included basal stem rot and the presence of sclerotia in rotted tissue were observed on E. fortunei in Kunshan city, Jiangsu province, China. Disease incidence was estimated at approximately 15 to 20%; meanwhile, approximately 30 to 40% of diseased plants died. The infected plants showed brown to dark stem necrosis near the base, leaf yellowing and wilting. White mycelia and white to dark reddish-brown sclerotia were observed at the base of the stem and rotten tissue. To isolate the causal organism, infected stem tissue and sclerotia collected from diseased plants in a median strip in Kunshan (31°23'40"N, 120°54'57"E) were disinfected with 70% ethanol for 2 to 3 sec, followed by 2 min in 5% NaClO, rinsed three times with sterile water, then plated on potato dextrose agar (PDA) medium, and incubated at 25°C. Isolated colonies were subcultured by needle tip transfer 3 days later. Isolates had white mycelia on PDA, with a radial growth rate of 15.2 to 18.7 mm/day. White and orange sclerotia were developed after 5 to 8 days and eventually turned dark reddish-brown. The sclerotia were globoid or irregular with surface markings (1.4 to 4.3 mm diam.; mean = 2.59 mm; n = 50) on PDA, and the average number of sclerotia produced per Petri dish ranged from 35 to 85 (mean = 52; n = 10). Microscopic observations found septal hyphae and clamp connections. These morphological features were identical to the description of Sclerotium delphinii (syn. Sclerotium rolfsii var. delphinii) (Mukherjee et al. 2015; Punja and Damiani 1996; Stevens 1931). A representative isolate YKY2020.01 was stored in the Key laboratory of National Forestry and Grassland Administration on Ecological Landscaping of challenging Urban Sites in Shanghai. For molecular identification, DNA of the isolate YKY2020.01 was extracted using the Fungal DNA Kit (OMEGA bio-tek, China). The internal transcribed spacer region (ITS fragment including ITS1, 5.8S rDNA, and ITS2 region) was amplified with primers ITS1/ITS4 (White et al. 1990), and then sequenced by Sangon Biotech (Shanghai, China). BLAST analysis in NCBI found the ITS sequence of YKY2020.01 (MW916955) was 99.84% similar to S. delphinii strain CBS272.30 (MH855140). Phylogenetic analysis using maximum likelihood (ML) method placed isolate YKY2020.01 in the same clade as S. delphinii. To evaluate pathogenicity, hyphal blocks (0.7 cm diam.) were placed at the base of the stem of healthy E. fortunei (n = 5 plants). Five healthy plants were inoculated by uncolonized agar blocks as controls. All plants were kept in a greenhouse with a temperature range from 21 to 25.6°C (mean = 24.9°C) and relative humidity of 50%. Inoculated plants were symptomatic after 3 days and wilted after 12 days. Symptoms in inoculated plants were similar to those observed under natural conditions, whereas the control group remained asymptomatic. The fungal pathogen was reisolated from symptomatic tissue and confirmed as S. delphinii. To the best of our knowledge, this is the first report of S. delphinii causing southern blight on E. fortunei in China and worldwide. This finding provides concise and practical information on the newly emerged disease of E. fortunei, which is beneficial for future disease management. References: Mukherjee, A. K., et al. 2015. J. Plant Pathol. 97:303. Punja, Z. K. and Damiani, A. 1996. Mycologia 88:694. Stevens, F. L. 1931. Mycologia 23:204. White, T. J., et al. 1990. Page 315 in PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA. Funding: This work was supported by the Key Project of Science and Technology Commission of Shanghai Municipality (19DZ1204102).

Plant Disease ◽  
2008 ◽  
Vol 92 (2) ◽  
pp. 318-318
Author(s):  
S. Zhao ◽  
G. Xie ◽  
H. Zhao ◽  
H. Li ◽  
C. Li

Snow lotus (Saussurea involucrata Karel. & Kir. ex Sch. Bip.) is an economically important medicinal herb increasingly grown in China in recent years. In June of 2005, a leaf spot disease on commercially grown plants was found in the QiTai Region, south of the Tianshan Mountain area of Xinjiang, China at 2,100 m above sea level. Disease incidence was approximately 60 to 70% of the plants during the 2006 and 2007 growing seasons. Initial symptoms appeared on older leaves as irregularly shaped, minute, dark brown-to-black spots, with yellow borders on the edge of the leaflet blade by July. As the disease progressed, the lesions expanded, causing the leaflets to turn brown, shrivel, and die. A fungus was consistently isolated from the margins of these lesions on potato dextrose agar. Fifty-eight isolates were obtained that produced abundant conidia in the dark. Conidia were usually solitary, rarely in chains of two, ellipsoid to obclavate, with 6 to 11 transverse and one longitudinal or oblique septum. Conidia measured 60 to 80 × 20 to 30 μm, including a filamentous beak (13 to 47 × 3.5 to 6 μm). According to the morphology, and when compared with the standard reference strains, the causal organism of leaf spot of snow lotus was identified as Alternaria carthami (1,4). Pathogenicity of the strains was tested on snow lotus seedlings at the six-leaf stage. The lower leaves of 20 plants were sprayed until runoff with conidial suspensions of 1 × 104 spores mL–1, and five plants sprayed with sterile distilled water served as controls. All plants were covered with a polyethylene bag, incubated at 25°C for 2 days, and subsequently transferred to a growth chamber at 25°C with a 16-h photoperiod. Light brown lesions developed within 10 days on leaflet margins in all inoculated plants. The pathogen was reisolated from inoculated leaves, and isolates were deposited at the Key Oasis Eco-agriculture Laboratory of Xinjiang Production and Construction Group, Xinjiang and the Institute of Biotechnology, Zhejiang University. No reports of a spot disease caused by A. carthami on snow lotus leaves have been found, although this pathogen has been reported on safflower in western Canada (3), Australia (2), India (1), and China (4). To our knowledge, this is the first report of a leaf spot caused by A. carthami on snow lotus in China. References: (1) S. Chowdhury. J. Indian Bot. Soc. 23:59, 1944. (2) J. A. G. Irwin. Aust. J. Exp. Agric. Anim. Husb. 16:921, 1976. (3) G. A. Petrie. Can. Plant Dis. Surv. 54:155, 1974. (4) T. Y. Zhang. J. Yunnan Agric. Univ.17:320, 2002.


Plant Disease ◽  
2003 ◽  
Vol 87 (2) ◽  
pp. 203-203
Author(s):  
S. T. Koike ◽  
S. A. Tjosvold ◽  
J. Z. Groenewald ◽  
P. W. Crous

Bells-of-Ireland (Moluccella laevis) (Lamiaceae) is an annual plant that is field planted in coastal California (Santa Cruz County) for commercial cutflower production. In 2001, a new leaf spot disease was found in these commercially grown cutflowers. The disease was most serious in the winter-grown crops in 2001 and 2002, with a few plantings having as much as 100% disease incidence. All other plantings that were surveyed during this time had at least 50% disease. Initial symptoms consisted of gray-green leaf spots. Spots were generally oval in shape, often delimited by the major leaf veins, and later turned tan. Lesions were apparent on both adaxial and abaxial sides of the leaves. A cercosporoid fungus having fasciculate conidiophores, which formed primarily on the abaxial leaf surface, was consistently associated with the spots. Based on morphology and its host, this fungus was initially considered to be Cercospora molucellae Bremer & Petr., which was previously reported on leaves of M. laevis in Turkey (1). However, sequence data obtained from the internal transcribed spacer region (ITS1, ITS2) and the 5.8S gene (STE-U 5110, 5111; GenBank Accession Nos. AY156918 and AY156919) indicated there were no base pair differences between the bells-of-Ireland isolates from California, our own reference isolates of C. apii, as well as GenBank sequences deposited as C. apii. Based on these data, the fungus was subsequently identified as C. apii sensu lato. Pathogenicity was confirmed by spraying a conidial suspension (1.0 × 105 conidia/ml) on leaves of potted bells-of-Ireland plants, incubating the plants in a dew chamber for 24 h, and maintaining them in a greenhouse (23 to 25°C). After 2 weeks, all inoculated plants developed leaf spots that were identical to those observed in the field. C. apii was again associated with all leaf spots. Control plants, which were treated with water, did not develop any symptoms. The test was repeated and the results were similar. To our knowledge this is the first report of C. apii as a pathogen of bells-of-Ireland in California. Reference: (1) C. Chupp. A Monograph of the Fungus Genus Cercospora. Cornell University Press, Ithaca, New York, 1954.


Plant Disease ◽  
2020 ◽  
Author(s):  
Boda Praveen ◽  
A. Nagaraja ◽  
M. K. Prasanna Kumar ◽  
Devanna Pramesh ◽  
K. B. Palanna ◽  
...  

Little millet (LM) is a minor cereal crop grown in the Indian sub-continent. During October 2018, dark brown, circular to oval necrotic spots surrounded by concentric rings were observed on the upper leaf surface of the LM (cv. VS-13) grown in the fields of the University of Agricultural Sciences, Bengaluru, India (13.0784oN, 77.5793oE). As the disease progressed, infected leaves became blighted. Disease incidence up to 53% was recorded in 3 fields of 0.4-hectare area each. Thirty symptomatic leaves were collected to isolate the associated causal organism. The margins of diseased tissue were cut into 5 × 5-mm pieces, surface-sterilized in 75% ethanol for 45 seconds followed by 1% sodium hypochlorite for 1 min, finally rinsed in sterile distilled water five times and placed on PDA. After 7 days of incubation at 25°C, greyish fungal colonies appeared on PDA. Single-spore isolations were performed to obtain ten isolates. Pure cultures of the fungus initially produced light gray aerial mycelia that later turned to dark grey. All isolates formed obclavate to pyriform conidia measured 22.66-48.97μm long and 6.55-13.79µm wide with 1-3 longitudinal and 2-7 transverse septa with a short beak (2.55-13.26µm) (n=50). Based on the conidial morphology, the fungus was identified as Alternaria sp. Further, the taxonomic identity of all ten isolates was confirmed as A. alternata using species-specific primers (AAF2/AAR3, Konstantinova et al. 2002) in a PCR assay. Later, one of the isolate UASB1 was selected, and its internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (gapdh), major allergen Alt a 1 (Alt a 1), major endo-polygalacturonase (endoPG), OPA10-2, and KOG1058 genes were amplified in PCR (White et al. 1990; Berbee et al. 1999; Woudenberg et al. 2015), and the resultant products were sequenced and deposited in the NCBI GenBank (ITS, MN919390; gapdh, MT637185; Alt a 1, MT882339; endoPG, MT882340; OPA10-2, MT882341; KOG1058, MT882342). Blastn analysis of ITS, gapdh, Alt a 1, endoPG, OPA10-2, KOG1058 gene sequences showed 99.62% (with AF347031), 97.36% (with AY278808), 99.58% (with AY563301), 99.10% (with JQ811978), 99.05% (with KP124632) and 99.23% (with KP125233) respectively, identity with reference strain CBS916.96 of A. alternata, confirming UASB1 isolate to be A. alternata. For pathogenicity assay, conidial suspension of UASB1 isolate was spray inoculated to ten healthy LM (cv. VS-13) plants (45 days old) maintained under protected conditions. The spore suspension was sprayed until runoff on healthy leaves, and ten healthy plants sprayed with sterile water served as controls. Later, all inoculated and control plants were covered with transparent polyethylene bags and were maintained in a greenhouse at 28±2 ◦C and 90% RH. The pathogenicity test was repeated three times. After 8 days post-inoculation, inoculated plants showed leaf blight symptoms as observed in the field, whereas no disease symptoms were observed on non-inoculated plants. Re-isolations were performed from inoculated plants, and the re-isolated pathogen was confirmed as A. alternata based on morphological and PCR assay (Konstantinova et al. 2002). No pathogens were isolated from control plants. There is an increasing acreage of LM crop in India, and this first report indicates the need for further studies on leaf blight management and the disease impacts on crop yields.


Plant Disease ◽  
2007 ◽  
Vol 91 (1) ◽  
pp. 109-109 ◽  
Author(s):  
X. M. Yang ◽  
J. H. Wang ◽  
S. P. Qu ◽  
L. H. Wang

Lily (Lilium spp.) is an economically important cut flower cultivated in China. The soilborne fungus, Sclerotium rolfsii, is a major pathogen on many plants. During July 2005, severe basal stem rot and bulb rot symptoms were observed on an oriental lily cultivar (Sorbonne) in some commercial fields in northern Kunming (China). Disease incidence ranged from 20 to 30% across fields. Leaves of infected plants were chlorotic initially. As the disease progressed, stems and bulbs rotted and plants wilted. In the presence of abundant moisture, a white mycelium occurred on infected tissues. White or light-to-dark brown sclerotia (1 to 3 mm in diameter) developed from mycelium. Fungal isolates from infected bulbs grown on potato dextrose agar (PDA) produced white mycelia and 1- to 2-mm diameter dark brown sclerotia. Sclerotia were nearly round with smooth surfaces and distributed over the entire colony. Isolates were identified as S. rolfsii on the basis of mycelial characteristics and color, size, and distribution of sclerotia. Pathogenicity was tested in the greenhouse on oriental lily cv. Sorbonne grown in pots (1 plant per pot, five replicates). Inoculum that consisted of 1 g per pot of wheat kernels infested with mycelium and sclerotia was placed at the base of each inoculated plant. Five noninoculated plants served as controls. The inoculation trial was repeated once. After inoculation, all plants were covered with a polyethylene bag for 72 h and kept at temperatures ranging between 25 and 27°C. Inoculated plants developed symptoms of leaf yellowing within 12 days, soon followed by the appearance of white mycelium and sclerotia, and then eventually wilted. Control plants remained symptomless. S. rolfsii was reisolated from inoculated plants. To our knowledge, this is the first report of southern blight caused by S. rolfsii on lily in China. Infection of lily bulbs by S. rolfsii may cause losses in production fields in China, and the presence of infected bulbs may also interfere with bulb shipment.


Plant Disease ◽  
2020 ◽  
Author(s):  
Jianqiang Zhang ◽  
Kangli Wu ◽  
Xiaomeng Zhang ◽  
Jiajia Li ◽  
Abdramane salah zene ◽  
...  

Celery (Apium graveolens) is one of the most widely grown vegetables in the world. A survey in Anding District of Gansu Province in 2019 showed that the incidence of celery leaf spot was 25%-45%. The disease mainly occurs in late June and July. The leaf spot is conducive to the onset at high temperature and humidity environment. The initial symptoms were many small light brown, irregular-shaped on the leaves. The lesions gradually enlarged in the later stage of the disease, and multiple lesions coalesced to form large irregular brown spots, eventually the whole leaves died. A 3~4mm leaf tissue was cut from the junction of the diseased leaf and the healthy area, the leaf tisse was surface-sterilized in 1.5% NaClO for 1 min and washed with sterile water. Then, it was incubated on potato dextrose agar (PDA) and obtained the pure culture (Q1). After 5 days of cultivation at 25°C, the fungal colonies were olivaceous to dark olive with white margins and abundant aerial mycelia. The conidia were obclavate or ellipsoid, pale brown, with 3~4 longitudinal septa and 2~7 transverse septa, and measured 20.0 to 50.0 × 3.5 to 14.0μm (n=50). Conidiophores were septate, arising singly, and measured 3.5 to 40.0 × 2.5 to 4.5 μm (n=50). Based on morphological characteristics, the fungus was preliminarily identified as A.tenuissima (Simmons 2007). To further confirm the identification, the internal transcribed spacer region (ITS), translation elongation factor 1-α gene (TEF), RNA polymerase II second largest subunit (RPB2), major allergen Alt a 1 gene (Alt a 1), endopolygalacturonase gene (endoPG), anonymous gene region (OPA10-2) and glyceraldehyde 3-phos-phatedehydrogenase (GAPDH) were amplified and sequenced using primers ITS1/ITS4 (Peever et al. 2004), EF1-728F/EF1-986R (Carbone et al. 1999), RPB2-5F2/RPB2-5R (Sung et al. 2007), Alt-for/Alt-rev (Hong et al. 2005), EPG-specific/EPG-3b (Peever et al. 2004), OPA10-2R/OPA10-2L (Peever et al. 2004) and Gpd1/Gpd2 (Berbee et al. 1999) (GenBank accession no.MN046364, MW016001, MW016002, MW016003, MW016004, MW016005, MW016006). DNA sequences of TEF, RPB2, endoPG, OPA10-2 and GAPDH were 100% identical to those of A. tenuissima (MN256108, MK605866, KP789503, JQ859829 and MK683802), but ITS and Alt a 1 were 100% similarity with A. tenuissima (MN615420, JQ282277) and A. alternate (MT626589, KP123847). The ITS and Alt a 1 sequence did not distinguish A. tenuissima from the A. alternate complex. Maximum likelihood phylogenetic analyses were performed for the combined data set with TEF, RPB2, and endoPG using MEGA6 under the Tamura-Nei model (Kumar et al. 2016). The isolate Q1 clustered with type strain A. tenuissima CBS 918.96. The 20 celery plants of 4-7 leaf age were used test the pathogenicity of Q1, the ten plants were sprayed with 20ml of spore suspension (1×105 spores/ml), the control was sprayed with 20mL sterile water, which were placed in a growth chamber (25℃, a 14h light and 10h dark period, RH > 80%). Eight days after inoculation, 40% of the leaves formed lesions, which were consistent with the field observation,the control group was asymptomatic. The pathogen was reisolated from infected leaves to fulfill Koch’s postulates. To our knowledge, this is the first report of A. tenuissima causing leaf spot on celery in China.


Plant Disease ◽  
2021 ◽  
Author(s):  
Sujata Singh Yadav ◽  
Priyanka Suryavanshi ◽  
Indrajeet Nishad ◽  
Soumya Sinha

Sweet basil (Ocimum basilicum L.; Family Lamiaceae) is an annual aromatic and medicinal plant grown in tropical and subtropical regions of the world. In India, it is cultivated as a commercial crop on ~8,000 ha. Aerial plant parts and essential oil of sweet basil are used in pharmaceutical, perfumery, food industries and in different formulations of traditional Ayurvedic and Unani medicines (Shahrajabian et al. 2020). The leaves have the highest concentrations of secondary metabolites such as terpenes and phenylpropanoids which provide the distinctive aroma (Viuda-Martos et al. 2011). During October 2020, severe foliar disease was observed in experimental fields of sweet basil at Council of Scientific and Industrial Research (CSIR)-Central Institute of Medicinal and Aromatic Plants (CIMAP) in Lucknow, India. Initial symptoms included large, interveinal chlorotic lesions on the adaxial surface of the leaves and black sporulation on the abaxial surface. Within a few days, the abaxial side of leaves turned necrotic, and leaf senescence and defoliation occurred on plants with severe symptoms. Disease incidence was 20 to 30% of plants. The pathogen was characterized morphologically using a light microscope. Sporangiophores were hyaline, dichotomously branched, 186.9 to 423.07 × 6.85 to 9.06 µm and, branched 3 to 5 times with each branch, terminating in two slightly curved branchlets, the longer one 7.05 to 25.31 µm and the shorter one 4.98 to 15.92 µm. Each branchlet had a single sporangium at the tip. Conidia were ellipsoidal to sub-globose, olive-brown in color, and typically measured 25.21 to 33.86 × 17.92 to 26.24 µm, each, without a pedicel. Based on these morphological characteristics, the foliar disease was identified as downy mildew was caused by Peronospora belbahrii (Thines et al. 2009). Eight symptomatic and two asymptomatic plant samples were collected from different locations in the field, and genomic DNA was extracted from the conidia of the eight naturally infected tissues of sweet basil samples as well as leaf tissues from two asymptomatic plants, using the CTAB method. The internal transcribed spacer region was amplified using ITS1 and ITS4 primers. Only eight infected samples amplified products of expected size (~ 700 bp) and two asymptomatic samples showed no amplification. Only five amplified PCR products were sequenced (White et al. 1990). All five sequences were identical and were a 98.1% match with five P. belbahrii isolates (MN450330.1, MN308051.1, MH620351.1, KJ960193, and MF693898). The consensus sequence was deposited into the NCBI database (GenBank Accession No. MW689257). Downy mildew caused by P. belbahrii previously has been reported on sweet basil from several countries (Wyenandt et al. 2015). To confirm the pathogenicity of these isolates on sweet basil (cv. CIM-Saumya), 25 - day-old sweet basil plants were sprayed with a suspension (1 × 105 sporangia/ml) of P. belbahrii. All plants were kept in a growth chamber with a 23/18°C diurnal cycle with 65 to 85% relative humidity for 24 h. Non-inoculated plants treated with sterile water served as a control treatment. After 8 days, typical symptoms of downy mildew appeared on all the inoculated plants while non-inoculated plants remained asymptomatic. Inoculated leaves with symptoms consistent of downy mildew were collected and the causal agent again identified as P. belbahrii on the basis of microscopic examination and ITS rDNA sequence data. To our knowledge, this is the first report of downy mildew caused by P. belbahrii on sweet basil in India. The pathogen has a broad host range and may pose a serious threat to the cultivation of this valuable crop in India. Thus, it is pertinent to develop effective control measures to avoid further spread and mitigate economic loss. References: Shahrajabian, M. H., et al. 2020. Int. J. Food Prop. 23:1961-1970. Wyenandt, C. A., et al. 2015. Phytopathology 105:885. Thines, M., et al. 2009. Mycol. Res. 113:532. White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Viuda-Martos, M., et al. 2011. Food Control. 22:1715.


Plant Disease ◽  
2013 ◽  
Vol 97 (8) ◽  
pp. 1114-1114 ◽  
Author(s):  
D. Zhao ◽  
Y. B. Kang

Tree peony (Paeonia suffruticosa Andrews) is a perennial woody deciduous shrub native to China and famous for its beautiful flowers. Starting in early autumn 2010, blighted branches of tree peony were detected in the International Peony Garden in Luoyang. The disease incidence was greater than 10% and disease symptoms included bulb atrophy and twig and branch dieback. Pycnidia were embedded within the bark of diseased branches. They were small, black, ostiolate, and measured 145 to 275 × 140 to 251 μm. Pycnoconidia were single-celled, hyaline or sandy beige, rounded to ellipsoidal, and 3.9 to 10.3 × 2.3 to 7.0 μm. Pure cultures were obtained by plating the pycnoconidia on potato dextrose agar (PDA). In culture, the fungus produced a circular, white to pink colony with pyknotic and linter shaped aerial mycelium. Numerous pycnidia, initially brown and dark at maturity, were embedded in the mycelium, especially in the center of the colony, with a few of them scattered in the edge. The morphological characteristics were consistent with Phoma (2). The ITS1-5.8S-ITS2 region of three isolates were PCR amplified and sequenced with primers ITS1 and ITS4. Sequences (GenBank Accession No. JX885584) showed 99% identity with reference isolates of Peyronellaea glomerata (Corda) Goid (AB470906.1 and HQ380779.1) and Phoma glomerata (Corda) Wollenw. & Hochapfel (EU098115.1). These two species are synonyms (1). To test pathogenicity, nine healthy branches of 3-year-old potted tree peony plants were wound-inoculated with a PDA disk containing pycnidia from an actively growing colony of P. glomerata. Three control branches were inoculated with sterile PDA disks. Each inoculated branch was wrapped in a plastic bag and maintained in a greenhouse at 25 to 28°C. After 3 days, brown patches appeared on inoculated branches and extended by up to 1 cm. Pycnidia identical to those observed in the field and in storage appeared on all inoculated branches 7 days after inoculation. Control branches did not show symptoms. The pathogen was reisolated from inoculated branches, fulfilling Koch's postulates. P. glomerata was reported as the causal agent of withering of flowers and young shoots of grapevines in Yugoslavia (3). To our knowledge, P. glomerata and Botryosphaeria dothidea have always been reported together, causing branch wilting or dieback. To our knowledge, this is the first report of branch blight of tree peony caused by P. glomerata in China. References: (1) M. M. Aveskamp et al. Mycol. Soc. Am. 101:363, 2009. (2) G. H. Boerema et al. Studies in Mycology, 3, 1973. (3) A. Šaric-Sabadoš et al. Atti Ist. bot. Univ. Pavia 18:101, 1960.


Plant Disease ◽  
2012 ◽  
Vol 96 (8) ◽  
pp. 1226-1226
Author(s):  
A. Nasehi ◽  
J. B. Kadir ◽  
M. A. Zainal Abidin ◽  
M. Y. Wong ◽  
F. Mahmodi

In June 2011, tomatoes (Solanum lycopersicum) in major growing areas of the Cameron Highlands and the Johor state in Malaysia were affected by a leaf spot disease. Disease incidence exceeded 80% in some severely infected regions. Symptoms on 50 observed plants initially appeared on leaves as small, brownish black specks, which later became grayish brown, angular lesions surrounded by a yellow border. As the lesions matured, the affected leaves dried up and became brittle and later developed cracks in the center of the lesions. A survey was performed in these growing areas and 27 isolates of the pathogen were isolated from the tomato leaves on potato carrot agar (PCA). The isolates were purified by the single spore technique and were transferred onto PCA and V8 agar media for conidiophore and conidia production under alternating light (8 hours per day) and darkness (16 hours per day) (4). Colonies on PCA and V8 agar exhibited grey mycelium and numerous conidia were formed at the terminal end of conidiophores. The conidiophores were up to 240 μm long. Conidia were oblong with 2 to 11 transverse and 1 to 6 longitudinal septa and were 24 to 69.6 μm long × 9.6 to 14.4 μm wide. The pathogen was identified as Stemphylium solani on the basis of morphological criteria (2). In addition, DNA was extracted and the internal transcribed spacer region (ITS) was amplified by universal primers ITS5 and ITS4 (1). The PCR product was purified by the commercial PCR purification kit and the purified PCR product sequenced. The resulting sequences were 100% identical to published S. solani sequences (GenBank Accestion Nos. AF203451 and HQ840713). The amplified ITS region was deposited with NCBI GenBank under Accession No. JQ657726. A representative isolate of the pathogen was inoculated on detached 45-day-old tomato leaves of Malaysian cultivar 152177-A for pathogenicity testing. One wounded and two nonwounded leaflets per leaf were used in this experiment. The leaves were wounded by applying pressure to leaf blades with the serrated edge of a forceps. A 20-μl drop of conidial suspension containing 105 conidia/ml was used to inoculate these leaves (3). The inoculated leaves were placed on moist filter paper in petri dishes and incubated for 48 h at 25°C. Control leaves were inoculated with sterilized distilled water. After 7 days, typical symptoms for S. solani similar to those observed in the farmers' fields developed on both wounded and nonwounded inoculated leaves, but not on noninoculated controls, and S. solani was consistently reisolated. To our knowledge, this is the first report of S. solani causing gray leaf spot of tomato in Malaysia. References: (1) M. P. S. Camara et al. Mycologia 94:660, 2002. (2) B. S. Kim et al. Plant Pathol. J. 15:348, 1999. (3) B. M. Pryor and T. J. Michailides. Phytopathology 92:406, 2002. (4) E. G. Simmons. CBS Biodiversity Series 6:775, 2007.


Plant Disease ◽  
2012 ◽  
Vol 96 (8) ◽  
pp. 1227-1227 ◽  
Author(s):  
A. Nasehi ◽  
J. B. Kadir ◽  
M. A. Zainal Abidin ◽  
M. Y. Wong ◽  
F. Abed Ashtiani

Symptoms of gray leaf spot were first observed in June 2011 on pepper (Capsicum annuum) plants cultivated in the Cameron Highlands and Johor State, the two main regions of pepper production in Malaysia (about 1,000 ha). Disease incidence exceeded 70% in severely infected fields and greenhouses. Symptoms initially appeared as tiny (average 1.3 mm in diameter), round, orange-brown spots on the leaves, with the center of each spot turning gray to white as the disease developed, and the margin of each spot remaining dark brown. A fungus was isolated consistently from the lesions using sections of symptomatic leaf tissue surface-sterilized in 1% NaOCl for 2 min, rinsed in sterile water, dried, and plated onto PDA and V8 agar media (3). After 7 days, the fungal colonies were gray, dematiaceous conidia had formed at the end of long conidiophores (19.2 to 33.6 × 12.0 to 21.6 μm), and the conidia typically had two to six transverse and one to four longitudinal septa. Fifteen isolates were identified as Stemphylium solani on the basis of morphological criteria described by Kim et al. (3). The universal primers ITS5 and ITS4 were used to amplify the internal transcribed spacer region (ITS1, 5.8, and ITS2) of ribosomal DNA (rDNA) of a representative isolate (2). A 570 bp fragment was amplified, purified, sequenced, and identified as S. solani using a BLAST search with 100% identity to the published ITS sequence of an S. solani isolate in GenBank (1). The sequence was deposited in GenBank (Accession No. JQ736024). Pathogenicity of the fungal isolate was tested by inoculating healthy pepper leaves of cv. 152177-A. A 20-μl drop of conidial suspension (105 spores/ml) was used to inoculate each of four detached, 45-day-old pepper leaves placed on moist filter papers in petri dishes (4). Four control leaves were inoculated similarly with sterilized, distilled water. The leaves were incubated at 25°C at 95% relative humidity for 7 days. Gray leaf spot symptoms similar to those observed on the original pepper plants began to develop on leaves inoculated with the fungus after 3 days, and S. solani was consistently reisolated from the leaves. Control leaves did not develop symptoms and the fungus was not reisolated from these leaves. Pathogenicity testing was repeated with the same results. To our knowledge, this is the first report of S. solani causing gray leaf spot on pepper in Malaysia. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) M. P. S. Camara et al. Mycologia 94:660, 2002. (3) B. S. Kim et al. Plant Pathol. J. 15:348, 1999. (4) B. M. Pryor and T. J. Michailides. Phytopathology 92:406, 2002.


2019 ◽  
Vol 8 (1) ◽  
pp. 29-30
Author(s):  
Salman Ghuffar ◽  
Gulshan Irshad ◽  
Amjad S. Gondal ◽  
Raees Ahmad ◽  
Hafizi B. Rosli ◽  
...  

During June 2016, a postharvest survey of table grapes was carried out in the main fruit markets of Attock (33°46'07.9"N 72°21'43.0"E) and Jehlum (32°56'22.3"N 73°43'31.4"E) districts of Punjab Province. At the time of sampling, two cultivars (King’s Ruby and Perlette) were sampled at five different locations of both districts. Disease incidence % of bunches averaged 3 to 4 %. Some infected fruit appeared water-soaked, light brown and covered by fluffy mycelium consisting of erect sporangiophores with black sporangia on the top (Figure 1). Symptomatic tissue pieces were surface-sterilized with 0.1% NaOCl for 30 sec, rinsed three times with sterile distilled water, dried on filter paper for 45 sec and incubated on potato dextrose agar (PDA) at 25°C. After 1 day, mycelium on PDA was transferred to a fresh PDA plate and incubated at 25°C with a 12-h photoperiod. Within 3 days, white to yellow colonies with black aerial sporangia were formed (Figure 2). A total of 64 isolates were examined morphologically. Sporangiophores were erect, light brown and 623 to 3800 µm long. One to three rhizoids were observed opposite to each sporangiophore. Sporangia were black, globose to sub-globose, 91 to 124 μm in diameter. Columellae were conical to cylindrical and 86 to 187 μm long × 72 to 205 μm wide. Sporangiospores were hyaline to light dark grey, globose, ellipsoidal in shape and 6.8 to 12.4 × 3.6 to 12.5 μm (Figure 3). These features were identical to the description of Rhizopus stolonifer (Ehrenb.) Vuill (Liou et al., 2007). For molecular identification, the ITS1-5.8S-ITS2 region of two representative isolates (Rizo 05 and Rizo 07) was amplified with primers ITS1/ITS4 (White et al., 1990). Sequence comparison of two isolates Rizo05 and Rizo 07 (Accession no. MH348205  and MH356272) revealed 100% identity with previously reported isolates of Rhizopus stolonifer (Accession no. MG865992, KU729185, HM051076, and MF374842). To complete Koch’s postulates, 10-µl aliquots of spore suspensions (106 spores/ml) of Rizo 05 and Rizo 07 were pipetted onto three non-wounded and four wounded asymptomatic grape berries (seven berries per isolate), Sterile distilled water was applied to asymptomatic berries to serve as a negative control. Berries were incubated at 25 ± 2°C in sterile moisture chambers, and the experiment was conducted twice (Ghuffar et al., 2018). Black to light brown, fluffy mycelium with the original symptoms was observed on both wounded and non-wounded inoculated berries after 3 days, whereas no symptoms were recorded on the negative control (Figure 4). The morphology of the fungus that was re-isolated from each of the inoculated berries was identical to that of the original cultures. Previously, Rhizopus stolonifer has been reported as a pathogen on grapes in Chile (Latorre et al., 2002). To our knowledge, this is the first report of Rhizopus stolonifer causing bunch rot of grapes in Pakistan. This finding will help to plan effective disease management strategies against Rhizopus rot of grapes in Pakistan.


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