First Report of Fusarium oxysporum f. sp. ciceris on chickpea (Cicer arietinum L.) in Serbia

Chickpea (Cicer arietinum L.) is one of the most commonly consumed legume crops worldwide, cultivated in more than 55 countries (FAOSTAT, 2017). However, in Serbia, it is a novel crop grown on approximately 120 acres, but the areas under this crop slightly increase each year. Fusarium wilt caused by F. oxysporum f. sp. ciceris is one of the most economically important disease in the most chickpea-growing areas (Jimenez-Diaz et al., 2015), but yet there has been no formal report of the Fusarium wilt of chickpea in Serbia. In June 2018, the first symptoms of Fusarium wilt were registered at Rimski Šančevi (Vojvodina Province), Serbia (N 45019.311' E 019049.933') as wilted chickpea plants grouped in patches on approximately 5% plants in the area of 3 acres. Symptoms as yellowing and necrosis of foliage appeared as late wilt in the podding stage. Roots of affected plants showed no external discoloration, but cross-sections showed dark-brown discoloration of xylem tissue. To isolate the causal agent, symptomatic plants were collected, and ten cuttings of symptomatic root tissue were surface disinfected with 2% sodium hypochlorite solution for 5 min, rinsed three times in sterile distilled water, air-dried on sterilized filter paper and plated on potato dextrose agar (PDA) and water agar (WA) amended with streptomycin sulfate. After seven days of incubation at 25°C in the dark, isolates were preliminarily identified according to their morphological characters and Fusarium Laboratory Manual (Leslie and Summerell, 2006). For each isolate 100, micro/macroconidia and chlamydospores were measured. Conidia were hyaline; macroconidia sickle-shaped, with blunt ends, 2-4 septa (10.1-17.7 x 3.1-5.8 µm); microconidia ellipsoidal, 0-1 septa (4.9-8.6-2.7-3.5 µm). Chlamydospores were globose (4.3-8.8 µm). Representative isolates (K343, K375 and K378) were purified by a single-spore technique for further analyses (Leslie and Summerell, 2006). Molecular identification of three representative isolates (K343, K375 and K378) was made by sequencing the rRNA internal transcribed spacer (ITS) region and translation elongation factor 1α (TEF1) gen. For all isolates, the ITS and TEF1 genes were amplified and sequenced with primers ITS1/4 (White et al. 1990), EF1-728 and EF1-986 (Rehner and Buckley 2005). Based on a BLAST search of the NCBI nucleotide database, the ITS sequences (GenBank MK920204.1, MK928423.1 and MK928424.1) had 99.8% identity with F. oxysporum f. sp. ciceris isolate (MK074845.1). The TEF1 (GenBank MN788462.1, MN788463.1 and MN788464.1) had 96.3 -100% identities with F. oxysporum f. sp. ciceris isolate (FJ538245.1). The pathogenicity test was conducted on 7-day old plants according to the drench method described by Maitlo et al. (2016). The concentration of inoculation suspensions was adjusted on 1x106, and ten plants per isolate were tested and inoculated with 10 ml of suspension. Control plants were drenched with 10 ml of sterilized distilled water. Nine days after inoculation, first symptoms as leaves wilting and white mycelia presence around stem base occurred on plants inoculated with isolate K378. On day 11, the first symptoms occurred on plants inoculated with isolates K343 and K375 as well. Up to the 13th day after inoculation, all plants were wilted, and the pathogen was successfully reisolated and confirmed as F. oxysporum f.sp. ciceris. To the best of our knowledge, this is the first report of F. oxysporum f.sp. ciceris causing Fusarium wilt on chickpea in Serbia. Acknowledgements This research was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia, grant number: 451-03-9/2021-14/200032 References: FAOSTAT. http://www.fao.org/faostat/en/#data/QC. Retrieved 20 February 2010. Jiménez-Díaz, R. M., et al. 2015. Crop Prot. 73: 16-27. Leslie, J. F., and Summerell, B. A. 2006. Page 26 in: The Fusarium Laboratory Manual. Blackwell, Ames, IA. Mailto, S. A., et al. 2016. Int. J. Agric. Biol. 18(1): 31-36. Rehner, S. A., and Buckley, E. 2005. Mycologia 97:84 White, T. J., et al. 1990. Pages 315-322 in: PCR Protocols. A Guide to Methods and Applications. Academic Press, San Diego, CA.

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First Report of Phaeoacremonium mortoniae Associated with Grapevine Decline in Iran

Plant Disease ◽

10.1094/pdis-05-11-0361 ◽

2011 ◽

Vol 95 (8) ◽

pp. 1034-1034 ◽

Cited By ~ 6

Author(s):

H. Mohammadi

Keyword(s):

Fungal Pathogens ◽

Vascular Tissue ◽

Sodium Hypochlorite Solution ◽

Malt Extract ◽

Fars Province ◽

Distilled Water ◽

Conidial Suspension ◽

Single Spore ◽

First Report ◽

Grapevine Decline

In July 2009, a survey was conducted in individually owned rooted vineyards in Iran to determine fungal pathogens associated with grapevine decline. Symptoms of grapevine decline such as slow dieback, stunted growth, small chlorotic leaves, and reduced foliage were observed on 7-year-old grapevines (cv. Askari) in Bavanat (Fars Province, southwestern Iran). Internal wood symptoms such as black spots and dark brown-to-black vascular streaking were observed in cross and longitudinal sections of stems and trunks. Wood samples were collected from symptomatic trunks and cordons. The bark of each fragment was removed and 10 thin cross sections (2 to 3 mm thick) were cut from symptomatic vascular tissue of the samples. These disks were immersed in 1.5% sodium hypochlorite solution for 4 min, washed thrice with sterile distilled water, and plated onto malt extract agar (MEA) supplemented with 100 mg liter–1 of streptomycin sulfate. Plates were incubated at 25°C in darkness. All colonies were transferred to potato dextrose agar (PDA) and incubated at 25°C. Five isolates of a Phaeoacremonium sp. were obtained. Single-spore isolates were transferred to PDA, MEA, and oatmeal agar (OA) media and incubated at 25°C for 8 to 16 days in the dark (2). Colonies reached a radius of 9.5 to 12 mm after 8 days of incubation. Colonies were flat and yellowish white on PDA and OA and white-to-pale gray after 16 days of incubation on MEA. Conidiophores were short and unbranched, 14 to 38.5 (23.5) μm long, and often ending in a single terminal phialide. Phialides were terminal or lateral and mostly monophialidic. Conidia were hyaline, oblong to ellipsoidal or reniform, 2 to 6.5 (4.9) μm long, and 1.1 to 1.7 (1.4) μm wide. On the basis of these characteristics, the isolates were identified as Phaeoacremonium mortoniae (1,2). Additionally, identity of the PMH1 isolate was confirmed by sequencing a fragment of the -tubulin gene with primers T1 and Bt2b (GenBank Accession No. JF831449). The sequence of this isolate was identical to the sequence of P. mortoniae (GenBank Accession No. HM116767). Pathogenicity tests were conducted on 2-month-old grapevine seedlings of cv. Askari by watering the roots with 25 ml of a conidial suspension (107 conidia ml–1) harvested from 21-day-old cultures grown on MEA. Controls were inoculated with 25 ml of sterile distilled water. Fifteen replicates were used for each isolate with an equal number of noninoculated plants. All plants were grown under greenhouse conditions (25 to 30°C). Two months after inoculation, inoculated seedlings showed reduced growth, chlorotic leaves, epinasty, severe defoliation, and finally wilting, while control seedlings remained healthy. The fungus was reisolated from internal tissues of the stems of inoculated seedlings. To my knowledge, this is the first report of P. mortoniae causing grapevine decline in Iran. References: (1) M. Groenewald et al. Mycol. Res. 105:651, 2001. (2) L. Mostert et al. Stud. Mycol. 54:1, 2006.

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First report of Fusarium falciforme (FSSC 3+4) causing wilt disease of Phaseolus vulgaris in Mexico

Plant Disease ◽

10.1094/pdis-06-20-1160-pdn ◽

2020 ◽

Author(s):

José Francisco Díaz-Nájera ◽

Sergio Ayvar-Serna ◽

Antonio Mena-Bahena ◽

Emiliano Baranda-Cruz ◽

Mateo Vargas-Hernández ◽

...

Keyword(s):

Phaseolus Vulgaris ◽

Tap Water ◽

Wilt Disease ◽

Healthy Plant ◽

Pathogenicity Test ◽

Distilled Water ◽

Conidial Suspension ◽

Single Spore ◽

First Report ◽

Fusarium Falciforme

Bean (Phaseolus vulgaris) is the second most important crop in Mexico after corn due to the high consumption of beans in all regions of the country. In the winter (January 2016), bean plants showing wilting, root discoloration and necrosis were observed, with an incidence of approximately 30% in different fields (<1 ha) in Tecoanapa, Guerrero State, Mexico. Symptomatic fine roots (<2 mm) were cut into 0.5 cm long pieces, washed with tap-water, surface disinfected with 1.5% NaOCl for 3 min, and rinsed with sterile distilled water. Thirty-five pieces were placed on potato dextrose agar (PDA, Difco) and incubated at 25 ℃ for seven days. Then, single-spore isolates were obtained. Colonies on PDA showed abundant white aerial mycelium and a growth rate of 4.5 mm/day, and in reverse, colonies were white/pink with a brown centre. Microconidia were cylindrical to ellipsoid, aseptate, hyaline and 7.8-(6.0)-4.7 × 2.7-(2.1)-1.6 µm. On carnation leaf agar, macroconidia were 37.8-(29.4)-23.5 × 4.1-(3.5)-2.6 µm, hyaline, falcate, with slightly curved apexes, and 3-5 septa. Chlamydospores were round, intercalary, hyaline, single or in chains (Boot 1971). A representative strain (CSAEGRO-AyDi-Ef) was analyzed by PCR and the translation elongation factor 1-alpha (tef1) gene (GenBank accession number MK945757) was sequenced using the EF-1/EF-2 primers (O’Donnell 2000). FUSARIUM-ID (Geiser et al. 2004) analysis showed 100% similarity with the Fusarium solani species complex (FSSC 3+4) strain NRRL28562. In addition, Bayesian phylogenetic analysis placed this strain in the Fusarium falciforme clade. A pathogenicity test was performed by immersing healthy plant roots (cv. Negro Jamapa) in 200 mL of a conidial suspension (50×106 conidia mL-1) for 10 min, and then transplanting the plants into pots. Control plants were immersed in sterile distilled water. Similar symptoms as those in the field were observed at 10 days after inoculation, and the controls were healthy. The fungus was reisolated from infected plants and showed the same morphology and tef1 sequence as the original isolate, fulfilling Koch’s postulates. Recently, F. falciforme was reported to cause wilting of P. vulgaris in Cuba (Duarte et al. 2019); however, this is the first report of F. falciforme (FSSC 3+4) causing wilt disease of P. vulgaris in Mexico. This species was previously reported in Mexico affecting onion (Tirado-Ramírez et al. 2018), papaya, tomato (Vega-Gutiérrez et al. 2019a, b), and maize (Douriet-Angulo et al. 2019), suggesting an ample host range in the country.

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First Report of Alternaria spp. Causing Leaf Spot on Sweet Viburnum in China

Plant Disease ◽

10.1094/pdis-06-20-1407-pdn ◽

2021 ◽

Author(s):

Chaodong Qiu ◽

Yingying Zhang ◽

Zhenyu Liu

Keyword(s):

Leaf Spot ◽

Genomic Dna ◽

Morphological Characteristics ◽

Leaf Spot Disease ◽

Molecular Characteristics ◽

Pathogenicity Test ◽

Distilled Water ◽

Single Spore ◽

First Report ◽

Sweet Viburnum

Sweet viburnum [Viburnum odoratissimum (L.) Ker Gawl] is an evergreen shrub mainly cultivated along roadsides in urban landscapes and also in parks and residential areas. A foliar disease occurred on about 40% of sweet viburnum plants near Anhui Grand Theatre, Anhui Province of China in June 2019. In early stages of sweet viburnum infection, the symptoms appeared as small brown spots ranged in length from 2 to 3 millimeters on the leaves. The spots developed on the upper, middle, and lower leaves of the plant, however, the upper leaves got more severely affected. As the disease develops, the spots enlarged and became rectangular or oval, brown to dark-brown, and their centers became ashen gray. In later stages of infection, the diseased leaves became wilting. Diseased leaves were surface disinfested and three small sections (2-3 mm2) were cut from the margin of the lesions. Sections were placed in 1.5% NaClO for 2 min, submerged in three changes of sterilized distilled water for 1 min each, placed onto potato dextrose agar (PDA) medium amended with 50 μg/ml of ampicillin and kanamycin, and incubated at 25℃ for 3 days. The mycelium from the leading edge of colonies growing from the tissue was sub-cultured onto a PDA plate for 3 days, followed by spore induction (Simmons 2007) and single spore isolation to obtain a pure culture of the putative pathogen. Colonies of one single spore isolate HF0719 were rounded, grayish white with dense aerial mycelium viewed from above and dark brown viewed from below. On potato carrot agar (PCA) medium, conidiophores were branched or occasionally unbranched. On branched conidiophores, conidia were in dwarf tree-like branched chains of 2-5 conidia. On unbranched conidiophores, conidia were simple or in chains of 2-8 conidia. Conidia were light brown or dark brown, ovoid, ellipsoidal to fusiform, and ranged in size from 7 to 26.5 × 4.5 to 11 μm with an average size of 16 × 7 µm based on 500 spore observations, with one beak and 1-7 transverse, 0-3 longitudinal, and 0-3 oblique septa. Beaks were ranged in (1.5-)2-10(-16) μm long. Based on cultural and morphological characteristics, isolate HF0719 was identified as Alternaria spp. (Simmons 2007). For molecular identification, total genomic DNA was isolated from mycelia collected from 7 day-old colonies of isolate HF0719 using the fungal genomic DNA extraction kit (Solarbio, Beijing, China). Fragments of five genes, including those encoding glyceraldehyde-3-phosphate dehydrogenase (gpd), plasma membrane ATPase, actin, calmodulin, and the Alternaria major allergen (Alt a1) regions of isolate HF0719 were amplified and sequenced using primer pairs gpd1/gpd2 (Berbee et al. 1999), ATPDF1/ATPDR1, ACTDF1/ACTDR1, CALDF1/CALDR1 (Lawrence et al. 2013), and Alt-for/Alt-rev (Hong et al. 2005), respectively. The obtained nucleotide sequences were deposited into GenBank as accession numbers: gpd, MT614365; ATPase, MT614364; actin, MT614363; calmodulin, MN706159; and Alt a1, MN304720. Phylogenetic tree using a maximum likelihood bootstrapping method based on the five-gene combined dataset in the following order: gpd, ATPase, actin, calmodulin, Alt a1 of HF0719 and standard strains representing 120 Alternaria species (Lawrence et al. 2013) was constructed. Isolate HF0719 formed a separate branch. On the basis of morphological characteristics and phylogenetic pattern, isolate HF0719 was identified as Alternaria spp.. A pathogenicity test was performed by rubbing 32 healthy leaves of six 5-year-old sweet viburnum plants with a cotton swab dipped in spore suspension containing 2.6 × 106 spores/ml, following leaf surface disinfection with 70% ethanol in the open field. Sterilized distilled water was used as control. The average air temperature was about 28℃ during the period of pathogenicity test. Eleven days after inoculation, 100% of inoculated leaves showed the leaf spot symptom identical to symptoms observed in the field. Control leaves were symptomless. The experiment was done three times. The re-isolated pathogen from the leaf lesion had the same morphological and molecular characteristics as isolate HF0719, thus satisfying Koch’s postulates. The genus Alternaria has been reported to cause leaf spot on sweet viburnum in Florida, USA (Alfieri et al. 1984). To our knowledge, this is the first report of Alternaria spp. causing leaf spot on sweet viburnum in China, a highly valued ornamental plant. Our findings will contribute to monitoring and adopting strategies for manage leaf spot disease on sweet viburnum.

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First report of Fusarium solani associated with the bulb rot of Tulip (Tulipa spp) in India

Plant Disease ◽

10.1094/pdis-05-21-0916-pdn ◽

2021 ◽

Author(s):

Qadrul Nisa ◽

Efath Shahnaz ◽

Saba Banday ◽

Ali Anwar ◽

Khalid Z Masoodi ◽

...

Keyword(s):

Fusarium Solani ◽

Indian Subcontinent ◽

Pathogenicity Test ◽

Distilled Water ◽

Conidial Suspension ◽

Single Spore ◽

First Report ◽

The World ◽

Bulb Rot ◽

Tulip Bulbs

Tulip is an ornamental bulbous flowering crop belonging to the Genus Tulipa and family Liliaceae. It is the first ranking bulbous ornamental plant in the world (Nayeem and Qayoom 2015). They are often the first flowers to witness the bloom in the spring. Kashmir valley is located in northern Himalayas in northwestern region of Indian subcontinent. It is the most alluring and fascinating place all over India and the home of famous “Indhra Gandhi Memorial Tulip garden”, the largest tulip garden in the entire Asia. However there are number of constraints in tulip cultivation among which bulb rot occupy a prominent place (Piwoni 2000). Bulb rot is posing problem to all the tulip growers throughout the world (De Hertogh et al. 1983). Rot symptoms were observed on tulip bulbs in field as well as in storage conditions (20-22◦C temperature with a relative humidity of 65%) in the summers of 2018 and 2019 in Shalimar fields of Kashmir. The main disease symptoms are yellow sunken spots on bulbs, purple-yellow coloration of leaves. Causal agent was isolated using tissue bit technique (Pathak 1972) on potato dextrose agar plates which where incubated at 24±2◦C . Single spore technique was used to obtain the pure isolate (Johnston and Booth 1983). The isolate covered the full plate (90mm) in ten days. The colony was dull whitish in color, flat and smooth with concentric ring formation in the culture plate with inner ring having a creamy exudation. The mycelium was septate, branched and hyaline in color and measured 3.50-5.20 µm in width with an average of 4.4 µm. Micro-conidia were hyaline, cylindrical to oval, 0-1 septa and measured 7.50-11.00×2.80-3.75 µm in size. Macro conidia were hyaline with 3-4 septa, fusiform, moderately curved which measured 21.15- 32.00×3.80-4.75 µm in size with an average of 28.50±0.21× 4.30±0.2 µm. On the basis of these morphological and cultural characteristics of the fungus, it was identified as Fusarium solani (Mar.) Sacc.,. To confirm the identity the PCR amplification was carried out for two genes Internal Transcribed Spacer (ITS 1, ITS 4)and Translation Elongation factor1-alpha gene (tef1- alpha) (O’Donnell et al. 1998; White et al. 1990). BLAST analysis of the sequence obtained for both the genes showed 99% homology with F. solani sequences in GenBank and Fusarium –ID databases. The sequences were deposited in the GenBank (Accession No MN611433, MW995477). Pathogenicity test was conducted on variety orange emperor both in laboratory and polyhouse. Bulbs were divided into three sets, (three bulbs per set) one set was given injury and dipped in conidial suspension (106 conidia/ml) for 30 min, another set was kept uninjured and dipped in spore suspension of same concentration, the third set was served as control and dipped in sterilized distilled water. All the respective sets were incubated in a moist chamber maintained at a temperature of 22 ◦C to observe symptoms. The injured ones showed symptoms after 7-8 days of inoculation, whereas the uninjured bulbs showed symptoms after 11-12 days. No symptoms were observed in controlled set. A pot experiment was also conducted to carry the pathogenicity tests. Bulbs were injured with the help of sterile needle and were dipped in conidial suspension (106 conidia/ml) for 30 min (Pastrana et al. 2014). The bulbs kept for control were dipped in sterilized distilled water. Bulbs were then planted in pots maintained at 18◦C. The above ground parts of the inoculated bulbs showed symptoms like stunted growth which gradually turned yellow and did not produced flowers. The bulbs after harvesting were rotten .No symptoms were observed in controlled plants. To fulfill the Koch's postulates the fungal pathogen was re-isolated which was identified as F. solani. The pathogen is reported to cause disease in other crops (Gupta et al. 2012) but to our knowledge and on the basis of literature, this is the first report of F. solani causing bulb rot of tulip in India.

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

Plant Disease ◽

10.1094/pdis-06-20-1371-pdn ◽

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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First Report of Seedborne Fusarium thapsinum and its Pathogenicity on Soybean (Glycine max) in the United States

Plant Disease ◽

10.1094/pdis-08-14-0806-pdn ◽

2014 ◽

Vol 98 (12) ◽

pp. 1745-1745 ◽

Cited By ~ 2

Author(s):

R. Pedrozo ◽

C. R. Little

Keyword(s):

United States ◽

The United States ◽

Soybean Plant ◽

Sodium Hypochlorite Solution ◽

Distilled Water ◽

Seedling Mortality ◽

Beta Tubulin ◽

First Report ◽

Significant Difference ◽

Germinated Seeds

A three-year survey from 2010 to 2012 was conducted in Kansas to investigate the identity and diversity of seedborne Fusarium spp. in soybean. A total of 408 soybean seed samples from 10 counties were tested. One hundred arbitrarily selected seeds from each sample were surface-sterilized for 10 min in a 1% sodium hypochlorite solution to avoid contaminants and promote the isolation of internal fusaria. Seeds were rinsed with sterile distilled water and dried overnight at room temperature (RT). Surface-sterilized seeds were plated on modified Nash-Snyder medium and incubated at 23 ± 2°C for 7 days. Fusarium isolates were single-spored and identified by morphological characteristics on carnation leaf agar (CLA) and potato dextrose agar (PDA) (3). From 276 seedborne Fusarium isolates, six were identified as F. thapsinum (2). On CLA, F. thapsinum isolates produced abundant mycelium and numerous chains of non-septate microconidia produced from monophialides. Microconidia were club-shaped and some were napiform. No chlamysdospores were found. On PDA, three of the isolates presented characteristic dark yellow pigmentation and three were light violet. Confirmation of the isolates to species was based on sequencing of an elongation factor gene (EF1-α) segment using primers EF1 and EF2 and the beta-tubulin gene using primers Beta1 and Beta2 (1). Sequence results (~680 bp, EF primers; ~600 bp, beta-tubulin primers) were confirmed by using the FUSARIUM-ID database (1). All isolates matched F. thapsinum for both genes sequenced (Accession No. FD01177) at 99% identity. Koch's postulates were completed for two isolates of F. thapsinum under greenhouse conditions. Soybean seeds (Asgrow AG3039) were imbibed with 2.5 × 105 conidia ml−1 for 48 h. After inoculation, seeds were dried for 48 h at RT. One isolate each of F. equiseti and F. oxysporum were used as the non-pathogenic and pathogenic inoculation controls, respectively. In addition, non-inoculated seeds and seeds imbibed in sterile distilled water (mock) were also used. Twenty-five seeds from each treatment were planted in pots (500 ml) with autoclaved soil and vermiculite (1:1). The experiment was a completely randomized design with three replicates (pots) per isolate. The entire experiment was repeated three times. After 21 days, aggressiveness of both F. thapsinum isolates was assessed using initial stand (%), final stand (%), and seed mortality (% of non-germinated seeds). Both seedborne F. thapsinum isolates caused reduced emergence and final stand, and increased seedling mortality when compared to the non-inoculated and F. equiseti controls (P< 0.0001). No significant difference was observed between F. thapsinum isolates and F. oxysporum. F. thapsinum isolates were re-isolated from wilted seedlings and non-germinated seeds, but not from the control treatments. Typically, F. thapsinum is considered a pathogen of sorghum, but it has also been recovered from bananas, peanuts, maize, and native grasses (3). However, its presence on soybean plant tissues and its pathogenicity has never been reported. To our knowledge, this is the first report of seedborne F. thapsinum and its pathogenicity on soybean in the United States. References: (1) D. M. Geiser et al. Eur. J. Plant Pathol. 110:473, 2004. (2) C. J. R. Klittich et al. Mycologia 89:644, 1997. (3) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Oxford, UK, 2006.

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First Report of Phytophthora drechsleri Associated with Stem and Foliar Blight of Gynura bicolor in Taiwan

Plant Disease ◽

10.1094/pdis-12-10-0931 ◽

2011 ◽

Vol 95 (7) ◽

pp. 874-874 ◽

Cited By ~ 1

Author(s):

Y. M. Shen ◽

C. H. Chao ◽

H. L. Liu

Keyword(s):

Disease Incidence ◽

Morphological Characteristics ◽

Hyphal Growth ◽

Pathogenicity Test ◽

Distilled Water ◽

First Report ◽

Foliar Blight ◽

Dual Cultures ◽

Phytophthora Drechsleri ◽

Gynura Bicolor

Gynura bicolor (Roxb. ex Willd.) DC., known as Okinawa spinach or hong-feng-cai, is a commonly consumed vegetable in Asian countries. In May 2010, plants with blight and wilt symptoms were observed in commercial vegetable farms in Changhua, Taiwan. Light brown-to-black blight lesions developed from the top of the stems to the petioles and extended to the base of the leaves. Severely infected plants declined and eventually died. Disease incidence was approximately 20%. Samples of symptomatic tissues were surface sterilized in 0.6% NaOCl and plated on water agar. A Phytophthora sp. was consistently isolated and further plated on 10% unclarified V8 juice agar, with daily radial growths of 7.6, 8.6, 5.7, and 2.4 mm at 25, 30, 35, and 37°C, respectively. Four replicates were measured for each temperature. No hyphal growth was observed at 39°C. Intercalary hyphal swellings and proliferating sporangia were produced in culture plates flooded with sterile distilled water. Sporangia were nonpapillate, obpyriform to ellipsoid, base tapered or rounded, and 43.3 (27.5 to 59.3) × 27.6 (18.5 to 36.3) μm. Clamydospores and oospores were not observed. Oospores were present in dual cultures with an isolate of P. nicotianae (p731) (1) A2 mating type, indicating that the isolate was heterothallic. A portion of the internal transcribed spacer sequence was deposited in GenBank (Accession No. HQ717146). The sequence was 99% identical to that of P. drechsleri SCRP232 (ATCC46724) (3), a type isolate of the species. The pathogen was identified as P. drechsleri Tucker based on temperature growth, morphological characteristics, and ITS sequence homology (3). To evaluate pathogenicity, the isolated P. drechsleri was inoculated on greenhouse-potted G. bicolor plants. Inoculum was obtained by grinding two dishes of the pathogen cultured on potato dextrose agar (PDA) with sterile distilled water in a blender. After filtering through a gauze layer, the filtrate was aliquoted to 240 ml. The inoculum (approximately 180 sporangia/ml) was sprayed on 24 plants of G. bicolor. An equal number of plants treated with sterile PDA processed in the same way served as controls. After 1 week, incubation at an average temperature of 29°C, blight and wilt symptoms similar to those observed in the fields appeared on 12 inoculated plants. The pathogen was reisolated from the lesions of diseased stems and leaves, fulfilling Koch's postulates. The controls remained symptomless. The pathogenicity test was repeated once with similar results. G. bicolor in Taiwan has been recorded to be infected by P. cryptogea (1,2), a species that resembles P. drechsleri. The recorded isolates of P. cryptogea did not have a maximal growth temperature at or above 35°C (1,2), a distinctive characteristic to discriminate between the two species (3). To our knowledge, this is the first report of P. drechsleri being associated with stem and foliar blight of G. bicolor. References: (1) P. J. Ann. Plant Pathol. Bull. 5:146, 1996. (2) H. H. Ho et al. The Genus Phytophthora in Taiwan. Institute of Botany, Academia Sinica, Taipei, 1995. (3) R. Mostowfizadeh-Ghalamfarsa et al. Fungal Biol. 114:325, 2010.

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First Report of Curvularia aeria on Etlingera linguiformis from Nagaland, India

Plant Disease ◽

10.1094/pdis-01-14-0060-pdn ◽

2014 ◽

Vol 98 (11) ◽

pp. 1580-1580 ◽

Cited By ~ 2

Author(s):

C. Kithan ◽

L. Daiho

Keyword(s):

Leaf Surface ◽

Agricultural Research ◽

Disease Incidence ◽

Indian Agricultural Research Institute ◽

Mountain Range ◽

Pathogenicity Test ◽

Distilled Water ◽

Conidial Suspension ◽

First Report ◽

Initial Symptoms

Etlingera linguiformis (Roxb.) R.M.Sm. of Zingiberaceae family is an important indigenous medicinal and aromatic plant of Nagaland, India, that grows well in warm climates with loamy soil rich in humus (1). The plant rhizome has medicinal benefits in treating sore throats, stomachache, rheumatism, and respiratory complaints, while its essential oil is used in perfumery. A severe disease incidence of leaf blight was observed on the foliar portion of E. linguiformis at the Patkai mountain range of northeast India in September 2012. Initial symptoms of the disease are small brown water soaked flecks appearing on the upper leaf surface with diameter ranging from 0.5 to 3 cm, which later coalesced to form dark brown lesions with a well-defined border. Lesions often merged to form large necrotic areas, covering more than 90% of the leaf surface, which contributed to plant death. The disease significantly reduces the number of functional leaves. As disease progresses, stems and rhizomes were also affected, reducing quality and yield. The diseased leaf tissues were surface sterilized with 0.2% sodium hypochlorite for 2 min followed by rinsing in sterile distilled water and transferred into potato dextrose agar (PDA) medium. After 3 days, the growing tips of the mycelium were transferred to PDA slants and incubated at 25 ± 2°C until conidia formation. Fungal colonies on PDA were dark gray to dark brown, usually zonate; stromata regularly and abundantly formed in culture. Conidia were straight to curved, ellipsoidal, 3-septate, rarely 4-septate, middle cells broad and darker than other two end cells, middle septum not median, smooth, 18 to 32 × 8 to 16 μm (mean 25.15 × 12.10 μm). Conidiophores were terminal and lateral on hyphae and stromata, simple or branched, straight or flexuous, often geniculate, septate, pale brown to brown, smooth, and up to 800 μm thick (2,3). Pathogen identification was performed by the Indian Type Culture Collection, Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi (ITCC Accession No. 7895.10). Further molecular identity of the pathogen was confirmed as Curvularia aeria by PCR amplification and sequencing of the internal transcribed spacer (ITS) regions of the ribosomal DNA by using primers ITS4 and ITS5 (4). The sequence was submitted to GenBank (Accession No. MTCC11875). BLAST analysis of the fungal sequence showed 100% nucleotide similarity with Cochliobolus lunatus and Curvularia aeria. Pathogenicity tests were performed by spraying with an aqueous conidial suspension (1 × 106 conidia /ml) on leaves of three healthy Etlingera plants. Three plants sprayed with sterile distilled water served as controls. The first foliar lesions developed on leaves 7 days after inoculation and after 10 to 12 days, 80% of the leaves were severely infected. Control plants remained healthy. The inoculated leaves developed similar blight symptoms to those observed on naturally infected leaves. C. aeria was re-isolated from the inoculated leaves, thus fulfilling Koch's postulates. The pathogenicity test was repeated twice. To our knowledge, this is the first report of the presence of C. aeria on E. linguiformis. References: (1) M. H. Arafat et al. Pharm. J. 16:33, 2013. (2) M. B. Ellis. Dematiaceous Hyphomycetes. CMI, Kew, Surrey, UK, 1971. (3) K. J. Martin and P. T. Rygiewicz. BMC Microbiol. 5:28, 2005. (4) C. V. Suberamanian. Proc. Indian Acad. Sci. 38:27, 1955.

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

Plant Disease ◽

10.1094/pdis-01-21-0223-pdn ◽

2021 ◽

Author(s):

Md Aktaruzzaman ◽

Tania Afroz ◽

Hyo-Won Choi ◽

Byung Sup Kim

Keyword(s):

Leaf Spot ◽

Ipomoea Batatas ◽

Rust Fungus ◽

Morphological Characteristics ◽

Perilla Frutescens ◽

Herbaceous Plant ◽

Pathogenicity Test ◽

Conidial Suspension ◽

Single Spore ◽

First Report

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

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