scholarly journals First Report of Leaf Spot of Rice Caused by Alternaria arborescens in Pakistan

Plant Disease ◽  
2014 ◽  
Vol 98 (6) ◽  
pp. 846-846 ◽  
Author(s):  
N. Akhtar ◽  
U. Bashir ◽  
S. Mushtaq
Keyword(s):  
Leaf Spot ◽  
Disease Incidence ◽  
Its Region ◽  
Rice Cultivar ◽  
Spore Suspension ◽  
Malt Extract ◽  
Fungal Culture ◽  
First Report ◽  
Morphological Studies ◽  
Alternaria Arborescens

Rice (Oryza sativa) is one of the most profitable and popular cereal crops in Pakistan. In July 2012, symptoms consisting of circular, black, necrotic spots, 2 to 4 mm in diameter, were observed on leaves of a commonly grown rice cultivar, Basmati-198, in private rice fields at Lahore (Punjab). This disease was observed later on rice cultivar KSK-133 grown at Faisalabad (Punjab) during the same cropping season. Disease incidence was ~35% and 25% for Basmati-198 and KSK-133, respectively. To our knowledge, the pathogen was confined only in these areas and cultivars and was not present on other rice varieties or crops. Ten infected plants were selected randomly from each field of two rice cultivars and one infected leaf for each of the 10 infected plants was selected for the isolation of fungal pathogen. Necrotic lesions were cut into pieces of ~2 mm2, surface-disinfected with 0.5% sodium hypochlorite, placed on 2% malt extract agar (MEA) (Sigma, Dorset, UK), and incubated at 25 ± 2°C for 4 to 5 days. Emerging fungal colonies were transferred aseptically to fresh MEA petri plates for purification. Alternaria spp. were consistently recovered from infected leaves. Three isolates per variety were selected for detailed morphological studies. Each isolate was grown at 25°C on MEA and potato carrot agar (PCA) for 7 days. All isolates displayed similar morphological features including black radiate, floccose colonies with irregular margins, 6 to 7 cm in diameter on MEA and 2 to 3 cm with 1 to 2 pairs of concentric growth rings on PCA. Conidial chains were not crowded with 1 to 10 conidia per branch and bearing several lateral branches. Conidiophores were tapering and narrow, 40 to 200 × 2 μm. Conidia were ovoid within a size range of 10 to 30 × 5 to 14 μm, with 1 to 5 transverse and 1 longitudinal septum. Conidial color darkens from a dull tan to a medium brown as the culture matures. Based on morphology, the pathogen was identified as Alternaria arborescens (1). A pure culture of the pathogen was deposited in First Fungal Culture Bank of Pakistan (FCBP) with accession FCBP1351. Identification based on morphology was verified by sequencing the internal transcribed spacer (ITS) region. For this, a DNA fragment of ~650 bp was amplified using total genomic DNA as template and ITS1 and ITS4 primers (2). The nucleotide sequence of the ITS region was submitted to GenBank under accession KF679683. Comparison of the sequence with those in GenBank revealed that the sequence was 99% identical with A. arborescens, isolate ALT-242 (KC415808), causing Eucalyptus leaf spot in India and strain STE-U4345 (AF404667), a causal pathogen of apple core rot in South Africa. Pathogenicity testing was performed on both cultivars. One-month-old plants grown in greenhouse were sprayed with 10 ml of spore suspension (2 × 105 spores/ml) as well as 10 ml of this spore suspension in soil at the time of sowing. Control plants were sprayed with sterilized water. The plants were covered with plastic bags for 48 h and kept under observation for 2 weeks in a glasshouse at 30 ± 2°C. Lesions appeared on leaves after 10 days of inoculation whereas control plants remained healthy. Pathogenicity tests were repeated in triplicate. Similar disease symptoms and re-isolation of A. arborescens fulfilled Koch's postulates. To our knowledge, this is the first report of A. arborescens leaf spot of rice in Pakistan. At present, the distribution of this disease is limited to the fields where it was observed. References: (1) E. G. Simmons. Alternaria: An Identification Manual. CBS, Fungal Biodiversity Center Utrecht, The Netherlands, 2007. (2) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

scholarly journals First Report of Rice Leaf Spot by Alternaria gaisen from Pakistan

Plant Disease ◽  
2014 ◽  
Vol 98 (10) ◽  
pp. 1440-1440
Author(s):  
N. Akhtar ◽  
R. Hafeez ◽  
Z. A. Awan
Keyword(s):  
Leaf Spot ◽  
Pure Culture ◽  
Spore Suspension ◽  
Sodium Hypochlorite Solution ◽  
Malt Extract ◽  
Fungal Culture ◽  
First Report ◽  
Rice Leaf ◽  
Morphological Studies ◽  
Necrotic Spots

A rice field owned by an individual grower in Lahore, Pakistan, was surveyed in July 2013. Plants with symptoms of black, circular, necrotic spots 3 to 4 mm in diameter and an average of 8 to 10 spots per leaf were observed. Diseased plants were present in the field either singly or in groups of three to five. Ten symptomatic plants were selected randomly, and one infected leaf per plant and one necrotic spot per leaf was selected for the isolation of the pathogen. Necrotic areas were cut into small pieces, surface sterilized with 1% sodium hypochlorite solution, and plated on 2% malt extract agar (MEA) (Sigma, Dorset, UK). After incubation at 25 ± 2°C for 4 to 5 days, fungal mycelium was transferred aseptically to fresh MEA for pure culture. Three different isolates grown for 7 days on MEA were selected for detailed morphological studies. The fungal colony was dark greenish-black, reaching 7 to 8 cm in diameter, with 2 to 3 poorly defined growth rings. Conidiophores were geniculate and 50 to 140 × 3 to 4.5 μm in size. Conidia were in chains of 4 to 10, ovoid, ranging in size from 35 to 50 × 8 to 10 μm, with 12 to 15 transversal and 0 to 2 longitudinal septa. Conidia darkened from dull tan yellow to brown as the culture aged. Based on morphological characteristics, the pathogen was identified as Alternaria gaisen (2). A pure culture of the pathogen was deposited in First Fungal Culture Bank of Pakistan (FCBP1354). Due to the complexity of morphology-based identification of the genus Alternaria, sequencing of the internal transcribed spacer (ITS) region was carried out using the ITS1/ITS4 primer pair (1,3). The nucleotide sequence (KJ806190) of an amplified DNA fragment was compared with those already submitted to GenBank. The BLAST results revealed 99% identity of our A. gaisen isolate to strains NW680 (EU520123.1), FC3s (JX391937.1), and CBS 632.93 (KC584197.1), as well as some other A. gaisen strains. Pathogenicity testing of the fungus was performed on Basmati-198, a common cultivar of rice in Pakistan, by either spraying leaves of 1-month-old plants with 10 ml of spore suspension (2 × 105 spores/ml) or mixing this spore suspension in soil at the time of sowing. Control plants were sprayed with sterilized water. Plants were kept in a glasshouse at 30 ± 2°C and monitored for disease development. After 15 days of incubation, similar leaf necrotic spots to those observed in the field, developed on all inoculated plants, whereas all control plants remained healthy and asympomatic. The experiment was repeated three times and similar results were obtained. Re-isolation of A. gaisen from the symptomatic leaves fulfilled Koch's pathogenicity postulate. Although limited to the field where it was observed, to our best of knowledge, this is the first report of rice leaf spot by A. gaisen from Pakistan. Also, rice has not been reported as the host of A. gaisen from any part of the world. This study indicates that A. gaisen is potentially an important pathogen of rice plants. Further investigations into epidemiology and disease management strategies for this new disease are warranted especially where rice crop is grown extensively. References: (1) G. S. de Hoog and R. Horre. Mycoses 45:259, 2002. (2) E. G. Simmons. Alternaria: An identification manual. CBS, Fungal Biodiversity Center Utrecht, The Netherlands, 2007. (3) T. J. White et al. Page 315 in: PCR Protocols, A Guide to Methods and Applications, Academic Press, San Diego, 1990.

scholarly journals First Report of Alternaria longipes Causing Leaf Spot of Potato Cultivar Sante in Pakistan

Plant Disease ◽  
2014 ◽  
Vol 98 (12) ◽  
pp. 1742-1742 ◽  
Author(s):  
A. Shoaib ◽  
N. Akhtar ◽  
S. Akhtar ◽  
R. Hafeez
Keyword(s):  
Leaf Spot ◽  
Potato Crop ◽  
Disease Incidence ◽  
Potato Cultivar ◽  
Leaf Spot Disease ◽  
Infected Leaf ◽  
Malt Extract ◽  
Fungal Culture ◽  
Causal Organism ◽  
First Report

Potato (Solanum tuberosum) is one of the most important vegetable crops worldwide, including Pakistan. During surveys from November to February of 2011 to 2013 in Sahiwal (Punjab), a severe leaf spot disease, new to farmers, was recorded. Symptoms consisted of 1- to 3-mm diameter black circular necrotic spots and appeared on the leaves of 2- to 3-week-old plants. Disease incidence was ~70 to 80%. This disease was localized to few fields in Sahiwal on potato variety Sante and to our knowledge, this has not been found on other areas or potato varieties in Pakistan. Fungi were isolated from randomly selected diseased plants. Ten infected plants were brought to the laboratory in sterilized polyethylene bags. One infected leaf per plant was selected for pathogen isolation. Infected parts of leaves were cut into ~2 mm2 pieces. Leaf pieces were surface sterilized for 1 min with 0.5% sodium hypochlorite and then inoculated aseptically onto 2% malt extract agar (MEA) (Sigma, Dorset, UK) and incubated at 25 ± 2°C for 3 to 4 days in the dark. Hyphal tip transfer from emerging colonies was performed to obtain pure cultures. Initial microscopic examination of pure fungal colonies revealed Alternaria as the likely causal organism. For morphology-based identification, five isolates from separate infected leaves were grown on MEA as well as potato carrot agar (PCA) for 7 days. All isolates showed similar morphological characters including dusty greenish black, floccose colonies with regular and smooth margins reaching 3 to 4.5 cm in diameter on MEA and sporulation with well-defined zones of growth. Aerial hyphae produced long branches that bore lateral chains of 1 to 7 conidia. Conidia were pointed at the tip, ovoid or ellipsoid, ranged from 18 to 40 × 5 to 12 μm with 4 to 8 transverse and 0 to 1 longitudinal septa. No conidial beak was present. Conidial color darkened from dull olive to brown as the culture matured. Based on morphology, the pathogen was identified as Alternaria longipes (1). A pure culture of a fungal pathogen was submitted to First Fungal Culture Bank of Pakistan (FCBP1355) for future reference. To confirm the morphology-based identification, the rDNA internal transcribed spacer (ITS) nucleotide sequence was amplified using ITS1 forward and ITS4 reverse primers (2). The amplicon of 537 bp was sequenced and submitted to GenBank under accession KJ806191. A BLASTn search using the KJ806191 sequence revealed it to be 99% identical to around 20 different strains of A. longipes deposited in GenBank including leaf spot pathogens of another Solanaceaeous member, Nicotiana tabacum (AY154684) and Asteraceous plant, Atractylodes macrocepha (JQ004404). Pathogenicity testing was performed in the greenhouse at 30 ± 2°C. Pots (16 × 9 cm) were filled with sterilized soil. Since spores of Alternaria sp. are known to survive in soil or plant debris, soil was sterilized and inoculated with 106 spore suspension of the isolated pathogen before sowing the potato seeds. Control pots were not inoculated. Approximately 10 days after plant germination, the previously observed disease symptoms appeared on leaves and A. longipes was re-isolated from the necrotic areas of leaves, thus fulfilling Koch's postulates. Plants in control treatments were asymptomatic. Pathogenicity tests were repeated three times. To our knowledge, this is the first report of A. longipes leaf spot of potato cultivar Sante from Pakistan. However, the distribution of this disease is confined to the area where it was observed, but it could be a threat for potato crop if not managed timely. References: (1) E. G. Simmons. Alternaria: An identification manual. CBS, Fungal Biodiversity Center Utrecht, The Netherlands, 2007. (2) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

scholarly journals First Report of Boeremia Blight Caused by Boeremia exigua var. exigua on Pyrethrum in Australia

Plant Disease ◽  
2011 ◽  
Vol 95 (11) ◽  
pp. 1478-1478 ◽  
Author(s):  
S. J. Jones ◽  
F. S. Hay ◽  
T. C. Harrington ◽  
S. J. Pethybridge
Keyword(s):  
Disease Incidence ◽  
Elongation Factor ◽  
Its Region ◽  
Tween 20 ◽  
Width Ratio ◽  
Malt Extract ◽  
Base Pairs ◽  
Internal Transcribed Spacer Region ◽  
First Report ◽  
Surface Sterilization

Pyrethrum (Tanacetum cinerariifolium) is produced for extraction of insecticidal compounds from the flower achenes. In 2004 and 2006, isolations from necrotic lesions on stems and leaves in three fields in northern Tasmania, Australia yielded four unidentified fungal isolates. Leaf lesions were medium brown and circular (2 to 4 mm in diameter) or irregular in shape (2 to 5 mm long). Stem lesions were irregular, necrotic spots, 5 to 15 mm below the flower peduncle, medium brown, 2 to 4 mm long, and 1 to 2 mm wide. Isolations were conducted on water agar following surface sterilization. Isolates were identified by colony characteristics and the presence of metabolite ‘E’ (1). On oatmeal agar (OA), colonies had irregular margins, were greenish olivaceous-to-olivaceous gray with sparse, white, floccose, aerial mycelia. On malt extract agar (MEA), cultures were variable in color with olivaceous black centers with soft, dense, aerial mycelia. Conidia were hyaline, ellipsoidal to oblong, mainly aseptate, but occasionally 1-septate with dimensions ranging from 2.5 to 7.5 × 1.8 to 3.8 μm (length/width ratio = 1.7 to 2.1). All isolates had moderate reactions to the NaOH test for metabolite ‘E’. DNA was extracted from all four isolates with a DNeasy Plant Mini Kit (QIAGEN Inc., Valencia, CA). For identification, the internal transcribed spacer region (ITS1, 5.8s, and ITS2) and part of the translation elongation factor (TEF) region were amplified and sequenced. Primers ITS1 and ITS4 (2) were used for the ITS region and primers EFCF1 (5′-AGTGCGGTGGTATCGACAAG) and EFCF6 (3′-CATGTCACGGACGGCGAAAC) were used for the TEF. Amplicons were sequenced in both directions and consensus sequences assembled. The ITS sequence was 100% identical to Boeremia exigua var. exigua (GenBank Accession No. GU237715). Base pairs 413 to 1,214 of the TEF sequence from the pyrethrum isolates matched base pairs 1 to 802 (799 of 802 identities) of B. exigua var. exigua (GenBank Accession No. GU349080). All isolates were confirmed as B. exigua var. exigua using morphology and sequencing. Pathogenicity tests were conducted three times in separate glasshouse trials for two of the four isolates. For each isolate, conidial suspensions in water (3 ml/plant) from MEA, adjusted to 5 × 105/ml were applied with Tween 20 (1 drop per 100 ml of water) to 8-week-old pyrethrum plants (five pots per isolate with four plants per pot) using a hand-held spray bottle. Twenty plants were sprayed with water and Tween 20 as nontreated controls. Plants were covered with plastic bags for 48 h after inoculation and examined for symptoms after 15 days at 20°C. Disease incidence (number of symptomatic leaves affected per total number of leaves) of the inoculated plants varied from 7.5 to 9.4%. Noninoculated plants did not develop symptoms. Isolations resulted in cultures morphologically identical on MEA and OA to those inoculated. To our knowledge, this is the first report of B. exigua var. exigua causing disease in pyrethrum. Cultures were deposited in the New South Wales Department of Agriculture collection (DAR79101 to 79104) and TEF and ITS sequences for DAR79101 in GenBank (Accession Nos. JF925328 and JF925329, respectively). Boeremia blight is likely to contribute to the fungal disease complex causing reductions in green leaf area in Australian pyrethrum production. References: (1) M. M. Aveskamp et al. Stud. Mycol. 65:1, 2010. (2) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

scholarly journals First Report of Soybean Rust Caused by Phakopsora pachyrhizi in Ghana

Plant Disease ◽  
2007 ◽  
Vol 91 (8) ◽  
pp. 1057-1057 ◽  
Author(s):  
R. Bandyopadhyay ◽  
P. S. Ojiambo ◽  
M. Twizeyimana ◽  
B. Asafo-Adjei ◽  
R. D. Frederick ◽  
...  
Keyword(s):  
Disease Incidence ◽  
Its Region ◽  
Spore Suspension ◽  
Soybean Rust ◽  
Plant Management ◽  
Phakopsora Pachyrhizi ◽  
Adaxial Side ◽  
First Report ◽  
Field Samples ◽  
Detached Leaves

Nigeria is the only country in West Africa where soybean rust, caused by Phakopsora pachyrhizi, has been officially reported (1). During a disease survey in Ghana during October 2006, soybean (Glycine max) leaves with rust symptoms (tan, angular lesions with erumpent sori exuding urediniospores) were observed in 11 fields in the following districts: Kassena Nankana in the Upper East Region; East Gonja, Central Gonja, and Tolon-Kumbungu in the Northern Region; and Ejisu-Juabeng in the Ashanti Region. Disease incidence in these fields ranged from 50 to 100% and disease severity ranged between 3 and 40% of the leaf area on infected plants. Urediniospores were hyaline, minutely echinulate, and 23 to 31 × 14 to 18 μm. Within a week of collection, leaf samples were sent to the USDA-ARS Foreign Disease-Weed Science Research Unit for verification of pathogen identity. DNA was extracted from leaf pieces containing sori with the Qiagen DNeasy Plant Mini kit (Valencia, CA), and all 11 field samples amplified in a real-time fluorescent PCR with the P. pachyrhizi-specific primers Ppm1 and Ppa2 (2). Sequence alignment of the internal transcribed spacer (ITS) region 2 further confirmed the identification as P. pachyrhizi (2). Infected leaves from three fields were separately washed in sterile water to collect urediniospores that were used to separately inoculate three detached leaves (for each isolate) of susceptible cultivar TGx 1485-1D (3). The abaxial surface of detached leaves was sprayed with 400 μl of spore suspension (1 × 106 spores per ml). A single leaf piece was placed in a 9-cm-diameter petri dish with adaxial side appressed on 1% technical agar amended with 10 μg/ml of kinetin. Lactic acid (1.5 ml/liter) and benomyl (12.5 mg/liter) were added to the agar medium to inhibit growth of saprophytic fungi and bacteria. Petri dishes were incubated at 20°C with a 12-h light/12-h dark cycle. Lesions on inoculated leaves developed 5 to 6 days after inoculation (DAI), and pustules (105 to 120 μm) formed 7 to 8 DAI and erupted 3 days later exuding columns of urediniospores similar in size to the initially collected isolates. Inoculating another set of detached leaves with a spore suspension (1 × 106 spores per ml) from the first set of detached leaves resulted in typical rust symptoms. The PCR assay, alignment of ITS region 2, morphological characters of the isolates, and pathogenicity tests demonstrate that P. pachyrhizi occurs in Ghana. To our knowledge, this is the first report of P. pachyrhizi in Ghana. References: (1) O. A. Akinsanmi et al. Plant Dis. 85:97, 2001. (2) R. D. Frederick et al. Phytopathology 92:217, 2002. (3) M. Twizeyimana et al. Online publication. http://www.plantmanagementnetwork.org/ infocenter/topic/soybeanrust/2006/posters/41.asp. Plant Management Network, 2006.

scholarly journals First Report of Corynespora Leaf Spot Caused by Corynespora cassiicola on Gynura in China

Plant Disease ◽  
2014 ◽  
Vol 98 (7) ◽  
pp. 1007-1007 ◽  
Author(s):  
B. J. Li ◽  
J. X. Chuan ◽  
M. Yang ◽  
G. F. Du
Keyword(s):  
Leaf Spot ◽  
Disease Incidence ◽  
Morphological Characteristics ◽  
Its Region ◽  
Leaf Spot Disease ◽  
Herbaceous Plant ◽  
Distilled Water ◽  
Corynespora Cassiicola ◽  
Single Spore ◽  
First Report

Gynura (Gynura bicolor DC.) is a perennial herbaceous plant in the family Compositae. It is an important Chinese vegetable, and is commonly used as a Chinese herbal medicine. In 2010, a severe leaf spot disease was observed on gynura grown in the main production areas in Tong Nan County, Chongqing City, China. Some farms experienced 60% disease incidence. Symptoms usually began on the lower leaves, as circular to elliptical or irregular spots with concentric rings. Individual spots were dark brown with grayish centers, sometimes coalescing and leading to extensive necrosis. The fungus associated with lesions was characterized as follows: Conidiophores were single or in clusters, straight or flexuous, unbranched, percurrent, cylindrical, pale to dark brown, 87.5 to 375.0 μm long and 5.0 to 10.5 μm wide. Conidia were solitary or catenate, straight to slightly curved, obclavate to cylindrical, 3 to 14 pseudoseptate, 82.8 to 237.5 μm long and 7.0 to 7.8 μm wide, and pale brown. The morphological characteristics of the conidia and conidiophores agreed with the descriptions for Corynespora cassiicola (1). To isolate the causal pathogen, surface-sterilized tissue at the margin of lesions was immersed in 75% ethanol for 30 s, rinsed in sterile water, dried in a laminar flow bench, transferred to PDA, and incubated at 28°C. Four single-spore cultures of the isolates were obtained and named from ZBTK10110637 to ZBTK10110640. All strains were identified as C. cassiicola. The isolate ZBTK10110637 was selected as representative for molecular identification. Genomic DNA was extracted by CTAB (2). The internal transcribed spacer (ITS) region of the rDNA was amplified using primers with ITS1 (5′-TCCGATGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′). Amplicons were 433 bp (GenBank Accession No. JX867272) and shared 100% similarity with that of C. cassiicola (NRC2-1 No. AB539285.1). To confirm pathogenicity, four isolates were used to inoculate 12 gynura plants (6 weeks old) by mist spray-inoculation with 108 spores/ml suspension in sterile distilled water on the leaves. Control plants were misted with sterile distilled water. After inoculation, all plants were incubated in a greenhouse maintained at 20 to 28°C with relative humidity of 80 to 85%. Five days after inoculation, dark brown spots with a grayish center typical of field symptoms were observed on all inoculated plants. No symptoms were seen on water-treated control plants. The fungus was re-isolated from inoculated plants. The morphological characteristics of isolates were identical with the pathogen recovered originally. This is the first report of C. cassiicola on gynura. References: (1) M. B. Ellis. CMI Mycological Papers 65(9):1-15, 1957. (2) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

scholarly journals First Report of a Leaf Spot on Pepper Caused by Cladosporium oxysporum in China

Plant Disease ◽  
2012 ◽  
Vol 96 (7) ◽  
pp. 1072-1072 ◽  
Author(s):  
X.-Y. Huang ◽  
Z.-H. Liu ◽  
J.-X. Hu ◽  
S.-W. Wang ◽  
Y. Zou ◽  
...  
Keyword(s):  
Leaf Spot ◽  
Morphological Characteristics ◽  
Its Region ◽  
Liaoning Province ◽  
Morphological Description ◽  
Hot Pepper ◽  
Malt Extract ◽  
First Report ◽  
Leaf Spots ◽  
Microscopic Morphology

Pepper (Capsicum annuum L.) is an important vegetable crop grown in Liaoning Province of China. In June 2009, leaf spotting was observed on hot pepper cv. 37-74 in Wafangdian County of Liaoning Province. By August 2011, the disease had spread to Pulandian and Donggang Counties. Symptoms initially appeared on both sides of leaves as pinpoint chlorotic spots that enlarged and developed into irregular, brown lesions, 1 to 8 mm in diameter. To identify the pathogen, leaf pieces (3 to 5 mm) taken at the edge of lesions with both infected and healthy portions were surface disinfected by placing them in 75% ethanol for 5 s, then transferred to a 0.1% aqueous mercuric chloride solution for 30 s and rinsed with sterilized water three times. The sections were placed on potato dextrose agar (PDA) at 25°C in the dark. Ten pure fungal cultures were obtained from single spores. For growth rate determination and morphological description of colonies, single conidial isolates were inoculated on PDA, malt extract agar (MEA), and oatmeal agar (OA), and incubated at 25°C for 14 days in darkness. For studies of microscopic morphology, isolates were grown on synthetic nutrient agar (SNA) in slide cultures. Colonies grew up to 77 to 80 mm in diameter on PDA, 62 to 66 mm on MEA, and 58 to 60 mm on OA after 14 days. Conidiophores were straight to slightly flexuous. Conidia ranged in shape from subglobose or ovoid to subcylindrical. Macronematous conidiophores measured 40 to 670 × 3 to 5.5 μm with swellings, micronematous conidiophores 15 to 137 × 1.5 to 3.5 μm, terminal conidia 2 to 4 × 2 to 3 μm, and intercalary conidia 4 to 10 × 2.5 to 4 μm. Ramoconidia were rarely observed. Secondary ramoconidia were zero- to one-septate and measured 5.2 to 14.8 × 2.4 to 4 μm. On the basis of these characteristics, the isolates were identified as Cladosporium oxysporum (1). The internal transcribed spacer (ITS) region from isolate W10-02 was amplified using primers ITS1 and ITS4 and sequenced (GenBank Accession No. JQ775499). The 525-bp amplicons had 99% identity to C. oxysporum (GenBank Accession No. EF029816). On the basis of morphological characteristics and nucleotide homology, the isolate was identified as C. oxysporum. Koch's postulates were fulfilled in the laboratory on pepper leaves inoculated with C. oxysporum conidial suspensions (107 conidia ml–1). Eight inoculated 4-week-old seedlings were incubated under moist conditions for 8 to 10 days at 25°C. All leaf spots that developed on inoculated leaves were similar in appearance to those observed on diseased pepper in the field. C. oxysporum was reisolated from lesions and its identity was confirmed by morphological characteristics. C. oxysporum was first reported as a pathogen of pepper in the Sultanate of Oman (2). To our knowledge, this is the first report of C. oxysporum causing leaf spot on pepper in China. The outbreak and spread of this disease may decrease the yield of pepper in the northern regions of China. More studies are needed on the management strategy including the resistance of pepper cultivars against C. oxysporum. References: (1) K. Bensch et al. 2010. Stud. Mycol. 67:1, 2010. (2) A. M. Hammouda. Plant Dis. 76:536, 1992.

scholarly journals First Report of Anthracnose on Camellia japonica Caused by Colletotrichum siamense in Zhejiang Province, China

Plant Disease ◽  
2021 ◽  
Author(s):  
Xiaojie Peng ◽  
Yuxuan Yuan ◽  
Shouke Zhang ◽  
Xudong Zhou
Keyword(s):  
Disease Incidence ◽  
Morphological Characteristics ◽  
Zhejiang Province ◽  
Internal Transcribed Spacers ◽  
Spore Suspension ◽  
Likelihood Method ◽  
Malt Extract ◽  
Camellia Japonica ◽  
First Report ◽  
Initial Symptoms

Camellia japonica is an attractive flowering woody plant with great ornamental and medicinal value in China. However, typical anthracnose lesions on the leaves are usually observed in summer in Zhejiang province. A number of 100 trees have been investigated with over 70% of leaf disease incidence. The symptom initially develops from the tip or edge of the leaf and dark green infected spots appear. The diseased spots expand and become yellow brown. The lesions are covered with abundant, small and black acervuli at the center with yellow edges. The diseased leaves become brittle, cracked, and finally fall off. Sixty leaves with typical anthracnose symptoms were sampled from gardens in Lin’an, Zhejiang province. The diseased tissues were cut into pieces and incubated in moist chambers at 25°C. The spore mass was collected using a sterile needle under dissection microscope and put on 2% malt extract agar (MEA). The cultures were incubated at 25°C in the dark for one week. Thirty single spore cultures were obtained and grown on 2% MEA at 25°C for morphological characterization. White aerial mycelia and black conidiomata with orange masses of conidia developed seven days later. Conidia are cylindrical in shape, 12-19 μm, mean lengths ranging from 15.5 ± 1.0 to 16.0 ± 1.2 μm. The morphological characteristics are consistent with those of Colletotrichum species. DNA was extracted from three selected isolates (HT-71, J-5, J-20) for sequencing. The partial regions of ribosomal internal transcribed spacers (ITS), calmodulin (CAL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin gene (ACT), beta-tubulin (TUB2), Apn2-Mat1-2 intergenic spacer and partial mating type gene (ApMat), and glutamine synthetase (GS) were amplified as described by Liu et al. (2015). Sequences of the above seven loci for the selected isolates were obtained, and deposited in the GenBank database (MZ014901 to MZ014905, MZ514915 to MZ514922, MZ514925 to MZ514930, MZ497332 and MZ497333). BLAST results indicate they represent Colletotricum siamense. Multi-locus phylogenetic analysis including ex-type of C. siamense (ICMP18578=CBS130417) and related species was conducted using Maximum Likelihood method, and C. acutatum (CBS 112996) served as the outgroup. The three obtained isolates clustered with the ex-type isolate of C. siamense. Eight leaves on two Camellia plants were inoculated to confirm the pathogenicity in the field. The leaves were surface sprayed with 75% ethanol and dried with sterilized filter paper. The leaves were inoculated using the wound/drop inoculation method: an aliquot of 10 μL of spore suspension (1.0 × 106 conidia per mL) was dropped on the left side of a leaf after wounding once by pin-pricking with a sterilized needle. The sterile water was dropped on the right side of the same leaf in parallel as control. The initial symptoms were observed seven days later, all inoculated leaves developed lesions similar to those observed in the field, and no symptoms observed in the control. The fungus was successfully re-isolated only from lesions inoculated with spore suspension exhibiting morphological characteristics resembling those in C. siamense, and further confirmed with sequence data. To our knowledge, this represents the first report of anthracnose on C. japonica caused by C. siamense worldwide. Confirmation of this pathogen in the region will be helpful for the disease management on C. japonica, considering previous report of C. camelliae-japonicae on the same host. References Fu, M., et al. 2019. Persoonia. 42: 1. https://doi.org/10.3767/persoonia.2019.42.01 Guarnaccia, V., et al. 2017. Persoonia. 39: 32. https://doi.org/10.3767/persoonia.2017.39.02 Hou, L. W., et al. 2016. Mycosphere. 7: 1111. Doi 10.5943/mycosphere/si/2c/4 Liu, F., et al. 2015. Persoonia. 35: 63. http://dx.doi.org/10.3767/003158515X687597 Vieira, A. D. S., et al. 2019. Mol. Phylogenet. Evol. https://doi.org/10.1016/j.ympev.2019.106694.

scholarly journals First Report of Gray Leaf Spot Caused by Pyricularia oryzae (synonym: Magnaporthe oryzae) in Oat (Avena sativa) in Georgia, USA.

Plant Disease ◽  
2021 ◽  
Author(s):  
Willis Turner Spratling ◽  
Suraj Sapkota ◽  
Brian Christopher Vermeer ◽  
Jason Mallard ◽  
Emran Ali ◽  
...  
Keyword(s):  
Avena Sativa ◽  
Leaf Spot ◽  
Pcr Amplification ◽  
Its Region ◽  
Gray Leaf Spot ◽  
Pyricularia Oryzae ◽  
Spore Suspension ◽  
Sterile Water ◽  
Economic Implications ◽  
First Report

In southeastern U.S., oat (Avena sativa L.) is predominantly grown as a grain or forage crop due to its exceptional palatability (Buntin et al. 2009). In November 2020, leaf spot symptoms were observed in an oat field (cv. Horizon 720) in Screven County, Georgia (GPS: 32°38'57.6"N 81°31'32.178"W). Lesions were oblong, whitish to gray in color, and surrounded by dark brown borders. Symptomatic oat leaves were sampled from the field and cut into 1 cm2 sections that were surface sterilized, plated onto Potato Dextrose Agar (PDA) media and incubated in the dark at 23°C. To obtain pure cultures, fungal hyphal tips were transferred onto fresh PDA plates 3 times. The pathogen was identified as Pyricularia (Magnaporthe) based on typical conidial morphology (Ellis 1971). Conidia were hyaline, pyriform, 2-septate, and displayed a basal hilum. Conidia measured 5.32 to 10.64 μm (average 8.24 μm) wide by 15.96 to 29.26 μm (average 25.40 μm) long. The identification of Pyricularia was further confirmed genetically via PCR amplification followed by sequencing. Genomic DNA was extracted from a 14-day old pure culture using a CTAB method (Doyle and Doyle 1987). The internal transcribed spacer (ITS) region of ribosomal DNA, calmodulin (CaM) gene, and -tubulin (TUB) gene were amplified using ITS5-ITS4 (White et al. 1990), CMD5-CMD6 (Hong et al. 2005), and Bt2a- Bt2b (Glass and Donaldson 1995) primer sets, respectively. Amplicons were Sanger sequenced and blasted against the NCBI database. Results exhibited 100% (ITS), 100% (CaM), and 99.61% (TUB) homology with Pyricularia oryzae Cavara (GenBank accession no. LC554423.1, CP050920.1, and CP050924.1, respectively). The ITS, CaM, and TUB sequences of the isolate were deposited in GenBank as MZ295207, MZ342893, and MZ342894, respectively. In a greenhouse (23°C, 80% RH), Koch’s postulates were carried out by using oat seedlings cv. Horizon 270 grown in Kord sheet pots filled with Sun Gro professional growing mix, and a P. oryzae spore suspension containing 104 conidia ml−1. The spore suspension (10 ml) was sprayed with an air sprayer onto 7 pots of oat seedlings at the two-leaf stage. Seven supplementary pots of oat seedlings of the same cultivar were sprayed with sterile water to act as controls. After inoculation, plants were covered with black plastic bags that had been sprayed with sterile water to maintain high humidity and incubated overnight in the greenhouse. The bags were removed the next day, and plants were evaluated for symptoms in the following days. Seven days after inoculation, plants displayed symptoms similar to those found in the original field sample. Control plants showed no symptoms. Pyricularia oryzae was consistently re-isolated from inoculated symptomatic oat tissues. To our knowledge, this is the first report of gray leaf spot caused by P. oryzae on oat in the state of Georgia and in the continental United States. Pyricularia oryzae can infect several graminaceous plants, including agronomically important crops such as rice (Oryza sativa) and wheat (Triticum spp.) (Chung et al. 2020). Phylogenetic analysis on the ITS region using 6 different host lineages was performed and revealed that this oat isolate was most closely related to the Lolium lineage. This outbreak could have economic implications in oat production.

scholarly journals First Report of Leaf Spot caused by Bipolaris oryzae on Switchgrass in Tennessee

Plant Disease ◽  
2013 ◽  
Vol 97 (12) ◽  
pp. 1654-1654 ◽  
Author(s):  
A. L. Vu ◽  
M. M. Dee ◽  
J. Zale ◽  
K. D. Gwinn ◽  
B. H. Ownley
Keyword(s):  
Leaf Spot ◽  
Panicum Virgatum ◽  
Morphological Characteristics ◽  
Its Region ◽  
Spore Production ◽  
Post Inoculation ◽  
Sterile Water ◽  
Bipolaris Oryzae ◽  
First Report ◽  
Symptomatic Leaf

Knowledge of pathogens in switchgrass, a potential biofuels crop, is limited. In December 2007, dark brown to black irregularly shaped foliar spots were observed on ‘Alamo’ switchgrass (Panicum virgatum L.) on the campus of the University of Tennessee. Symptomatic leaf samples were surface-sterilized (95% ethanol, 1 min; 20% commercial bleach, 3 min; 95% ethanol, 1 min), rinsed in sterile water, air-dried, and plated on 2% water agar amended with 3.45 mg fenpropathrin/liter (Danitol 2.4 EC, Valent Chemical, Walnut Creek, CA) and 10 mg/liter rifampicin (Sigma-Aldrich, St. Louis, MO). A sparsely sporulating, dematiaceous mitosporic fungus was observed. Fungal plugs were transferred to surface-sterilized detached ‘Alamo’ leaves on sterile filter paper in a moist chamber to increase spore production. Conidia were ovate, oblong, mostly straight to slightly curved, and light to olive-brown with 3 to 10 septa. Conidial dimensions were 12.5 to 17 × 27.5 to 95 (average 14.5 × 72) μm. Conidiophores were light brown, single, multiseptate, and geniculate. Conidial production was polytretic. Morphological characteristics and disease symptoms were similar to those described for Bipolaris oryzae (Breda de Haan) Shoemaker (2). Disease assays were done with 6-week-old ‘Alamo’ switchgrass grown from seed scarified with 60% sulfuric acid and surface-sterilized in 50% bleach. Nine 9 × 9-cm square pots with approximately 20 plants per pot were inoculated with a mycelial slurry (due to low spore production) prepared from cultures grown on potato dextrose agar for 7 days. Cultures were flooded with sterile water and rubbed gently to loosen mycelium. Two additional pots were inoculated with sterile water and subjected to the same conditions to serve as controls. Plants were exposed to high humidity by enclosure in a plastic bag for 72 h. Bags were removed, and plants were incubated at 25/20°C with 50 to 60% relative humidity. During the disease assay, plants were kept in a growth chamber with a 12-h photoperiod of fluorescent and incandescent lighting. Foliar leaf spot symptoms appeared 5 to 14 days post-inoculation for eight of nine replicates. Control plants had no symptoms. Symptomatic leaf tissue was processed and plated as described above. The original fungal isolate and the pathogen recovered in the disease assay were identified using internal transcribed spacer (ITS) region sequences. The ITS region of rDNA was amplified with PCR and primer pairs ITS4 and ITS5 (4). PCR amplicons of 553 bp were sequenced, and sequences from the original isolate and the reisolated pathogen were identical (GenBank Accession No. JQ237248). The sequence had 100% nucleotide identity to B. oryzae from switchgrass in Mississippi (GU222690, GU222691, GU222692, and GU222693) and New York (JF693908). Leaf spot caused by B. oryzae on switchgrass has also been described in North Dakota (1) and was seedborne in Mississippi (3). To our knowledge, this is the first report of B. oryzae from switchgrass in Tennessee. References: (1) D. F. Farr and A. Y. Rossman. Fungal Databases. Systematic Mycology and Microbiology Laboratory, ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/, 28 June 2012. (2) J. M. Krupinsky et al. Can. J. Plant Pathol. 26:371, 2004. (3) M. Tomaso-Peterson and C. J. Balbalian. Plant Dis. 94:643, 2010. (4) T. J. White et al. Pages 315-322 in: PCR Protocols: a Guide to Methods and Applications. M. A. Innis et al. (eds), Acad. Press, San Diego, 1990.

scholarly journals First report of Colletotrichum siamense Causing Blossom Blight on Thai basil (Ocimum basilicum L.) in Malaysia

Plant Disease ◽  
2020 ◽  
Author(s):  
Siti Izera Ismail ◽  
Nur Adlina Rahim ◽  
Dzarifah Zulperi
Keyword(s):  
Disease Incidence ◽  
Its Region ◽  
Ocimum Basilicum ◽  
Distilled Water ◽  
Single Spore ◽  
First Report ◽  
Plastic Bags ◽  
Colletotrichum Siamense ◽  
Primer Sets ◽  
Acca Sellowiana

Thai basil (Ocimum basilicum L.) is widely cultivated in Malaysia and commonly used for culinary purposes. In March 2019, necrotic lesions were observed on the inflorescences of Thai basil plants with a disease incidence of 60% in Organic Edible Garden Unit, Faculty of Agriculture in the Serdang district (2°59'05.5"N 101°43'59.5"E) of Selangor province, Malaysia. Symptoms appeared as sudden, extensive brown spotting on the inflorescences of Thai basil that coalesced and rapidly expanded to cover the entire inflorescences. Diseased tissues (4×4 mm) were cut from the infected lesions, surface disinfected with 0.5% NaOCl for 1 min, rinsed three times with sterile distilled water, placed onto potato dextrose agar (PDA) plates and incubated at 25°C under 12-h photoperiod for 5 days. A total of 8 single-spore isolates were obtained from all sampled inflorescence tissues. The fungal colonies appeared white, turned grayish black with age and pale yellow on the reverse side. Conidia were one-celled, hyaline, subcylindrical with rounded end and 3 to 4 μm (width) and 13 to 15 μm (length) in size. For fungal identification to species level, genomic DNA of representative isolate (isolate C) was extracted using DNeasy Plant Mini Kit (Qiagen, USA). Internal transcribed spacer (ITS) region, calmodulin (CAL), actin (ACT), and chitin synthase-1 (CHS-1) were amplified using ITS5/ITS4 (White et al. 1990), CL1C/CL2C (Weir et al. 2012), ACT-512F/783R, and CHS-79F/CHS-345R primer sets (Carbone and Kohn 1999), respectively. A BLAST nucleotide search of ITS, CHS-1, CAL and ACT sequences showed 100% similarity to Colletotrichum siamense ex-type cultures strain C1315.2 (GenBank accession nos. ITS: JX010171 and CHS-1: JX009865) and isolate BPDI2 (CAL: FJ917505, ACT: FJ907423). The ITS, CHS-1, CAL and ACT sequences were deposited in GenBank as accession numbers MT571330, MW192791, MW192792 and MW140016. Pathogenicity was confirmed by spraying a spore suspension (1×106 spores/ml) of 7-day-old culture of isolate C onto 10 healthy inflorescences on five healthy Thai basil plants. Ten infloresences from an additional five control plants were only sprayed with sterile distilled water and the inoculated plants were covered with plastic bags for 2 days and maintained in a greenhouse at 28 ± 1°C, 98% relative humidity with a photoperiod of 12-h. Blossom blight symptoms resembling those observed in the field developed after 7 days on all inoculated inflorescences, while inflorescences on control plants remained asymptomatic. The experiment was repeated twice. C. siamense was successfully re-isolated from the infected inflorescences fulfilling Koch’s postulates. C. siamense has been reported causing blossom blight of Uraria in India (Srivastava et al. 2017), anthracnose on dragon fruit in India and fruits of Acca sellowiana in Brazil (Abirami et al. 2019; Fantinel et al. 2017). This pathogen can cause a serious threat to cultivation of Thai basil and there is currently no effective disease management strategy to control this disease. To our knowledge, this is the first report of blossom blight caused by C. siamense on Thai basil in Malaysia.

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