First record of Ceratocystis fimbriata causing quick wilt of Calotropis gigantea in Pakistan

Calotropis gigantea belongs to the family Asclepiadaceae, native to Asia and commonly known as crown flower. C. gigantea was identified as an important medicinal plant in Asia and also harvested to obtain the fibres from the stem (Ganeshan et al. 2018; Narayanasamy et al. 2020). In April 2021, a quick wilt epidemic of C. gigantea was observed in District Gujranwala (32°05'58.0"N 74°02'38.0"E) Punjab, Pakistan. The wilt symptoms were very severe on mature plants with 60% disease incidence. Affected plants exhibited yellowing and quick wilting of leaves on each branch that eventually spread to the whole plant (Figure 1). Black patches were observed on the stem bark and cross section of infected stem showed black streaks in the xylem. The samples (n=33) were collected from symptomatic plants. Infected tissues were excised into 4-5mm segments, surface disinfested with 1% NaClO and rinsed 3 times with sterilized distilled water. To induce the sporulation of the fungus, the described carrot baiting method of Moller et al., (1968) was used. Single ascoma from carrot baits were picked under a microscope and transferred to malt extract agar (MEA) medium and incubated at 25°C. Purified cultures were obtained by single spore culture method and all the isolates were preserved with 35% glycerol at -80°C. The fungal colony was greyish olive with overripe banana odour. Hyphae were smooth and segmented. The perithecia were globose, black, measuring 190.27 to 257.34 × 186.11 to 243.24 µm, and showed a long black neck (732 to 977.34 µm). Cylindrical conidia (9.46 to 22.97 µm × 2.7 to 5.41 µm), barrel-shaped conidia (6.34 to 7.95× 8.15-10.41 µm) and chlamydospores (7.65-11.38µm×10.17-16.81µm) were observed (Figure 2). The isolates were identified as Ceratocystis fimbriata based on morphology and similar results were reported by Engelbrecht et al. (2005) and Suwandi et al. (2021). Genomic DNA was extracted from all isolates and multi-locus sequence analysis approach was used for molecular identification. The Internal Transcribed Spacers (ITS) region, the Translation Elongation Factor 1-α gene (TEF) and β-tubulin gene (TUB) were amplified using ITS1/4 (White et al. 1990), EF1-728F/ EF1-986R (Carbone et at. 1999) and βt1a/βt1b (Glass and Donaldson, 1995). Based on the BLAST analysis, all isolates were identified as C. fimbriata. The sequences of the representative isolate AK-W17 were submitted to the GenBank, NCBI database with the accession numbers (ITS:MZ711226), (TEF: MZ714595) and (TUB:MZ714596) showing 100% similarity with AF395687(ITS), MG980731(TEF) and AY177227(TUB) accessions of C. fimbriata. Based on Sequences similarity, representative isolate AK-W17 grouped with the isolates which representing C. fimbriata. Pathogenicity test was conducted on healthy C. gigantea seedlings grown in pots. A fungal mycelium plug (4×4mm) from 15 days old culture of AK-W17 was inserted into a slit (5 × 8 mm and made by puncturing the bark surface) in the stem bark, and the artificial wound was covered with wetted tissue paper and secured with Parafilm to maintain humidity, while control treatment was inoculated with only MEA medium plug. The seedlings were incubated under 70% relative humidity (RH) and 28°C in greenhouse. After 24 days, the inoculated seedlings showed 100% wilting identical to the original plant from which they were isolated (Figure 3). The control treatment had no symptoms. The pathogenic fungus was reisolated and identifies as C. fimbriata based on morphological and molecular characterization. C. fimbriata has been also reported to cause wilt disease in Dalbergia sissoo (Harrington et al. 2015) and pomegranate (Alam et al. 2017) in Pakistan. To our knowledge, this is the first report of C. fimbriata to cause C. gigantea quick wilt in Pakistan. C. fimbriata is one of the most aggressive plant pathogens and rapidly spread worldwide, so it is critical to implement appropriate management practices to reduce the fungus attack on plants.

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First Report of Wilt of Rubber Tree Caused by Chalaropsis thielavioides in China

Plant Disease ◽

10.1094/pdis-09-20-2066-pdn ◽

2020 ◽

Author(s):

Xue Li ◽

Jie Li ◽

Hua Yong Bai ◽

Kecheng Xu ◽

Ruiqi Zhang ◽

...

Keyword(s):

Rubber Tree ◽

Morphological Characteristics ◽

Spore Suspension ◽

Malt Extract ◽

Pathogenicity Test ◽

Distilled Water ◽

Ceratocystis Fimbriata ◽

First Report ◽

Mould Fungus ◽

Its Sequence Analysis

Rubber tree (Hevea brasiliensis (Willd. ex Adr. Juss) Müll. Arg.) is used for the extraction of natural rubber and is an economically and socially important estate crop commodity in many Asian countries such as Indonesia, Malaysia, Thailand, India, Sri Lanka, China and several countries in Africa (Pu et al, 2007). Xishuangbanna City and Wenshan City are the main rubber cultivation areas in Yunnan Province, China. In November 2012, rubber tree showing typical wilt symptoms (Fig. 1 A) and vascular stains (Fig. 1 B) were found in Mengla County, Xishuangbanna City. This disease was destructive in these trees and plant wilt death rate reached 5%. The diseased wood pieces (0.5cm long) from trunk of rubber was surface disinfected with 75% ethanol for 30s and 0.1% mercuric chloride (HgCl2) for 2min, rinsed three times with sterile distilled water, plated onto malt extract agar medium (MEA), and incubated at 28℃. After 7 days, fungal-like filaments were growing from the diseased trunk. Six cultures from 6 rubber trunk were obtained and incubated on MEA at 28℃, after 7 days to observe the cultural features. The mycelium of each culture was white initially on MEA, and then became dark green. Cylindrical endoconidia apices rounded, non-septate, smooth, single or borne in chains (8.9 to 23.6 × 3.81 to 6.3μm) (Fig. 1 C). Chlamydospores (Fig. 1 D) were abundant, thick walled, smooth, forming singly or in chains (11.1 to 19.2 × 9.4 to 12.0μm). The mould fungus was identifed as Chalaropsis based on morphology (Paulin-Mahady et al. 2002). PCR amplification was carried out for 3 isolates, using rDNA internal transcribed spacer (ITS) primer pairs ITS1F and ITS4 (Thorpe et al. 2005). The nucleotide sequences were deposited in the GenBank data base and used in a Blast search of GenBank. Blast analysis of sequenced isolates XJm8-2-6, XJm8-2 and XJm10-2-6 (accessions KJ511486, KJ511487, KJ511489 respectively) had 99% identity to Ch. thielavioides strains hy (KF356186) and C1630 (AF275491). Thus the pathogen was identified as Ch. thielavioides based on morphological characteristics and rDNA-ITS sequence analysis. Pathogenicity test of the isolate (XJm8-2) was conducted on five 1-year-old rubber seedlings. The soil of 5 rubber seedlings was inoculated by drenching with 40 ml spore suspension (106 spores / ml). Five control seedlings were inoculated with 40 ml of sterile distilled water. All the seedlings were maintained in a controlled greenhouse at 25°C and watered weekly. After inoculated 6 weeks, all the seedlings with spore suspension produced wilt symptoms, as disease progressed, inoculated leaves withered (Fig. 1 E) and vascular stains (Fig. 1 F) by 4 months. While control seedlings inoculated with sterile distilled water remained healthy. The pathogen re-isolated from all inoculated symptomatic trunk was identical to the isolates by morphology and ITS analysis. But no pathogen was isolated from the control seedlings. The pathogenicity assay showed that Ch. thielavioides was pathogenic to rubber trees. Blight caused on rubber tree by Ceratocystis fimbriata previously in Brazil (Valdetaro et al. 2015), and wilt by Ch. thielavioides was not reported. The asexual states of most species in Ceratocystis are “chalara” or “thielaviopsis” (de Beer et al. 2014). To our knowledge, this is the first report of this fungus causing wilt of rubber in China. The spread of this disease may pose a threat to rubber production in China.

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Ginger wilt and rot disease caused by Ceratocystis fimbriata in China

Plant Disease ◽

10.1094/pdis-08-20-1647-pdn ◽

2020 ◽

Author(s):

Ruiqi Zhang ◽

Kecheng Xu ◽

Xue Li ◽

Yang Gao ◽

Yuexian Sun ◽

...

Keyword(s):

Yunnan Province ◽

Disease Incidence ◽

Internal Transcribed Spacers ◽

Therapeutic Drug ◽

Malt Extract ◽

Sterile Water ◽

Ceratocystis Fimbriata ◽

Single Spore ◽

Initial Symptoms ◽

Rot Disease

Ginger (Zingiber officinale Rosc.) is an herb that has been grown in China for more than 2500 years. It can be used as both a spice and a therapeutic drug. In July 2013, ginger plants were found to have wilting symptoms and yellowing leaves with netrotics leaf tips in a farm in Kunming city of Yunnan province (25. 02 N; 102.42 E), southwest China, and we also found gray-black lesion on the surface of the harvest gingers in a market in Kunming. Initial symptoms on harvest gingers appeared as gray-black mycelia growth on the surface of the harvested ginger, which enlarged and extended internally. Carrot baiting was used to isolate the pathogen from rotted gingers and diseased ginger leaves (Moller and Devay. 1968). After two weeks, spores developing from perithecia on the carrot pieces were transferred to malt extract agar (MEA) and incubate at 25°C constant-temperature incubator. Six single-spore isolates (ZOR-1 to ZOR-6) were obtained, the isolates were stored in 15% glycerol at -80°C refrigerator in State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan Agricultural University. Cultures varied in color from white to brownish green to brown. N = 50 for all measurements. Blackish brown, globose perithecia (131.9 to 186.0 μm × 138.5 to 188.3 μm) with a long black neck (400.2 to 644.7 μm) were immersed, partially embedded or superficial on the substrate. Ascospores were globose or had a “hat-like” morphology typical of Ceratocystis fimbriata, and were 4.0 to 5.3 μm × 4.8 to 6.2 μm. Endoconidia were cylindrical and clavate (2.9 to 7.4 μm × 7.5 to 32.8 μm), conidia were barrel-shaped (4.4 to 10.4 μm× 6.2 to 12.9 μm), and chlamydospores were smooth, blackish brown, ovoid or obpyriform (8.42 to 12.21 μm × 10.47 to 17.65 μm) (Webster and Butler. 1967; Engelbrecht and Harrington. 2005). Genomic DNA was extracted from two isolates (ZOR-1, ZOR-2) using the CTAB method (Lee and Taylor 1990). The internal transcribed spacers (ITS) region of rDNA was amplified using primers ITS1F/ITS4 (Thorpe et al. 2005). The nucleotide sequences of ZOR-1 and ZOR-2 (GenBank accessions KJ511490 and KJ511491) were 100% homologous to those of the isolates of C. fimbriata from diseased Cucumis sativus L. and Punica granatum L. in China (GenBank accessions MH535909 and KT963159). Thus, the pathogen was identified as C. fimbriata. Pathogenicity tests were made on fresh ginger rhizomes in laboratory, the pathogen was cultured for 14 days on MEA (ZOR-1, ZOR-2), which were washed with sterilized water and the resulting spore suspensions diluted to 1.0 × 106 spores/ml . Wounds (0.5 × 0.5 cm) were made on the surface of healthy mature ginger rhizomes by scraping with a sterile scalpel, then treated with a 100 ul spore suspension. Control ginger rhizomes were coated sterile water. Ginger rhizomes were stored at room temperature. Each treatment was performed in triplicate. After 5 days, grey-black mycelia developed on the rhizome surface, becoming a visible black mould after 1 week. We reisolated the pathogen from infected tissues, but not from the controls. In the greenhouse, 20ml of 1.0 × 106 spores/ml suspensions from isolates ZOR-1 and ZOR-2, or sterile water were injected into two-month- old ginger seedlings in triplicate. The inoculated site on the stem turned black in 5 days. 6 weeks after inoculation, the inoculated plants developed yellowing leaves and wilting symptoms. The same fungus was re-isolated from inoculated plants, but not from the controls. According to Koch’s Postulation, the inoculated strains of ZOR-1 and ZOR-2 were the pathogens causing ginger wilt and rot disease. To the best of our knowledge, ginger is a new host plant of Ceratocystis fimbriata from China. In recent years, we have found that this disease incidence was approxmiatelt 5 to 10% of the farmland and 5 to 15% of the stored condition respectively in Yunnan Province. If not prevented ginger production in China will be affected.

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First Report of Fusarium culmorum and Microdochium bolleyi Causing Root Rot on Triticale in Kazakhstan

Plant Disease ◽

10.1094/pdis-12-20-2659-pdn ◽

2021 ◽

Author(s):

Mehtap Alkan ◽

Göksel Özer ◽

İmren Mustafa ◽

Fatih OZDEMIR ◽

Alexei Morgounov ◽

...

Keyword(s):

Root Rot ◽

Disease Incidence ◽

Fusarium Culmorum ◽

Gaeumannomyces Graminis ◽

Control Treatment ◽

Sodium Hypochlorite Solution ◽

Pathogenicity Test ◽

First Report ◽

Agar Plug ◽

Microdochium Bolleyi

Triticale (×Triticosecale Wittmack) is obtained from wheat × rye crossing. It is positioned between wheat and rye in terms of resistance to soilborne pathogens including Gaeumannomyces graminis var. tritici, Fusarium culmorum, F. avenaceum, and Bipolaris sorokiniana (Arseniuk and Góral 2015). In 2019, seven triticale fields were surveyed in Almaty Province, Kazakhstan to examine soil-borne fungal pathogens. A total of 28 symptomatic plants with stunting, rot or discolored root were collected to identify causal agents. The overall disease incidence was approximately 8 to 10% in the fields. Fungi were isolated from 3-5 mm pieces excised from symptomatic tissues. The pieces were exposed to surface disinfection in 1% sodium hypochlorite solution for 2 min, rinsed three times with sterile distilled water, blotted dry, and plated on 1/5 strength potato dextrose agar (PDA) amended with 0.01% streptomycin. Plates were left in the dark at 23°C for 7 days. A total of 34 fungal colonies were isolated of which nineteen isolates, originally from six fields showed the cultural characteristics of B. sorokiniana. This species was previously reported to cause common root rot on triticale in Kazakhstan (Özer et al. 2020). Ten isolates from four fields produced pale orange and cottony mycelium with red pigmentation on the agar, which is typical of Fusarium-like growth. The remaining isolates (n=5) from two fields produced salmon-colored and scarce aerial mycelium with no soluble pigmentation, similar to Microdochium spp. Fusarium isolates produced thick-walled and curved macroconidia with 3-4 septa (n=50, 25.7 to 37.6 × 4.1 to 7.3 μm in size) and notched basal cell on PDA, but microconidia were absent, which matches the description of F. culmorum (Wm.G. Sm.) Sacc. (Leslie and Summerell 2006). Microdochium isolates produced swollen, brown, and thick-walled chlamydospores and hyaline, one-celled, and thin-walled conidia (n=50, 5.4 to 9.3 × 1.5 to 3.0 μm in size) formed on ampullate and cylindrical conidiogenous cells on oatmeal agar (OA). These morphological features are consistent with previous observations for Microdochium bolleyi (R. Sprague) de Hoog & Herm.-Nijh. (Hong et al. 2008). To confirm morphological preliminary identifications, the portion of the translation elongation factor 1-alpha (EF1-α) gene was amplified with EF1/EF2 primers (O’Donnell et al. 1998) for representative Fusarium isolates (n=4) for each field. Additionally, the internal transcribed spacer (ITS) of ribosomal DNA was amplified with ITS1/ITS4 primers (White et al. 1990) for representative Microdochium isolates (n=2) for each field. BLASTn queries against NCBI GenBank revealed that the EF1-α sequences of Fusarium isolates (MW311081-MW311084) shared 100% identity with F. culmorum strain CBS 110262 (KT008433). The ITS sequences of M. bolleyi isolates (MW301448-MW301449) matched that of M. bolleyi strain CBS 137.64 (AM502264) with 100% sequence similarity. Pathogenicity test was conducted on pregerminated seeds of triticale cv. Balausa. A plastic pot (17 cm height, 9 cm in diam) was filled with a sterile mixture of vermiculite, peat, and soil (1:1:1, v/v/v). Mycelial plugs (1 cm in diam) were cut from the margin of a growing culture of representative isolates (Kaz_Fus123 and Kaz_Mb01) and placed onto the mixture in the pot. A sterile agar plug was employed as a control treatment. One pregerminated seed was put on the plug and covered with the mixture. The pots were transferred to a growth chamber set at 23 ± 2°C and a photoperiod of 14 hours. The experiment was performed twice using 5 replication pots per isolate. Four weeks after inoculation, discoloration of the crown was observed on all the inoculated roots, whereas no symptoms were observed on the control plants. Koch’s postulates were fulfilled by reisolating and identifying the pathogen based on the morphology described above. This is the first report of M. bolleyi and F. culmorum causing root rot on triticale in Kazakhstan. Although B. sorokiniana is the most primary pathogen that may limit yield in the production of triticale in Kazakhstan, F. culmorum and M. bolleyi have been found to be less frequent and less aggressive pathogens, respectively. Further studies are needed to better understand the potential distribution and impact of these pathogens on triticale.

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First Report of Leaf Spot Caused by Cladosporium oxysporum on Greenhouse Eggplant in China

Plant Disease ◽

10.1094/pdis-06-13-0606-pdn ◽

2014 ◽

Vol 98 (4) ◽

pp. 566-566 ◽

Cited By ~ 3

Author(s):

C. Zheng ◽

Z.-H. Liu ◽

S.-S. Tang ◽

D. Lu ◽

X.-Y. Huang

Keyword(s):

Leaf Spot ◽

Early Stage ◽

Disease Incidence ◽

Solanum Melongena ◽

Morphological Characteristics ◽

Liaoning Province ◽

Control Treatment ◽

Pathogenicity Test ◽

Potato Dextrose Broth ◽

First Report

Eggplant (Solanum melongena L.) is an important vegetable crop that has significant economic value in northern regions of China, especially in Liaoning Province. In April 2013, a leaf spot was discovered on the eggplant cultivar 706 in ten 1-ha commercial greenhouses in Huludao, Liaoning Province, with 30% of the eggplants infected, resulting in reduced eggplant yield and quality. By July 2013, disease incidence was 35%. Spots were found mainly on the leaves. At the early stage of infection, small, chlorotic spots appeared on leaves and gradually expanded into brown, irregular spots with a diameter of 1 to 7 mm. Dark green mold developed in the spots on both sides of the leaves at high humidity, and the spots led to leaf yellowing and defoliation. Conidiophores in the lesions were straight or slightly flexuous with 1 to 7 septa, brown and smooth, with typical swellings at the junction of septa, and 45 to 670 × 3.0 to 5.3 μm. Conidia were oval or obpyriform with a smooth surface, brown or dark brown, with 0 to 2 septa and 5.5 to 14.8 × 2.5 to 4.0 μm. The pathogen was consistent morphologically with Cladosporium oxysporum (1). To identify the pathogen, leaf pieces (3 to 5 mm2) taken from the edge of lesions so that each leaf section included both infected and healthy leaf tissue, were surface-disinfested in 75% ethanol for 30 s, then transferred to a 0.1% aqueous mercuric chloride solution for 30 to 60 s, and rinsed with sterilized water three times. The sections were cultured on potato dextrose agar (PDA) at 25°C in the dark for 7 days. Three pure cultures were obtained from single spores. The conidia on PDA were oval or obpyriform, and 5.4 to 14.7 × 2.4 to 4.2 μm with 0 to 1 septa, and were smaller than the conidia examined directly from infected eggplant leaves. Two isolates were grown on synthetic nutrient agar (SNA) in slide cultures. The conidiophores on SNA were straight or slightly flexuous with swellings at the junctions of septa. On the grounds of these morphological characteristics, the pathogen was identified as C. oxysporum (1,3). For DNA extraction, cultures were grown in potato dextrose broth and the internal transcribed spacer (ITS) region of ribosomal DNA (rNDA) was amplified using primers ITS1 and ITS4 (2). Sequence analysis showed that the ITS sequences of the two isolates were 99% identical to that of C. oxysporum (GenBank Accession No. EF029816). Two isolates were tested for pathogenicity on eggplant using 1 × 107 conidia/ml in sterilized water atomized onto each of six 7-week-old plants of the cultivar Xi'an Green Eggplant. Sterilized water was applied similarly to another six plants as the control treatment. The plants were incubated at 25°C with 85% relative humidity for 8 to 10 days. After 10 days, light brown, irregular spots were found on inoculated leaves, whereas no symptoms were observed on control plants. The pathogen was re-isolated from lesions on inoculated plants but not from control plants. The re-isolates were confirmed to be C. oxysporum based on morphological characteristics. The pathogenicity test was repeated and the same results obtained. Therefore, the pathogen causing leaf spot on eggplant in these greenhouses was identified as C. oxysporum. This is the first report of C. oxysporum causing leaf spot on greenhouse eggplant in China. C. oxysporum is a known pathogen of pepper and tomato. Additional studies are needed to provide management recommendations for this pathogen on Solanaceae crops. References: (1) K. Bensch et al. Stud. Mycol. 67:1, 2010. (2) Q. Li and G. Wang. Microbiol. Res. 164:233, 2009. (3) W. T. H. Peregrine and K. B. Ahmad. Phytopathol. Pap. 27:1, 1982.

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First Report of Coffee Canker Disease Caused by Ceratocystis fimbriata in China

Plant Disease ◽

10.1094/pdis-08-21-1808-pdn ◽

2022 ◽

Author(s):

Kecheng Xu ◽

Ruiqi Zhang ◽

Haixia Lu ◽

Jinglong Zhang ◽

Jing Yang ◽

...

Keyword(s):

Disease Incidence ◽

Morphological Characteristics ◽

Molecular Characteristics ◽

Outer Cell ◽

Leaf Margin ◽

Malt Extract ◽

Distilled Water ◽

Ceratocystis Fimbriata ◽

Canker Disease ◽

First Report

Coffee (Coffea arabica L.) is one of the most important agricultural commodities in the world market. As an important cash crop in China, coffee is cultivated mainly in Yunnan and Hainan provinces. During October 2013 and September 2020, coffee trees showing typical dieback and wilt symptoms were found in Nanping town (N 22° 38', E 101° 0'), Pu’er, and Puwen town (N 22° 32', E 101° 4'), Xishuangbanna in Yunnan province, China. Symptomatic trees initially exhibited yellowing of foliage, expanding in size along the leaf margin, then became blighted and dry, and the internal xylem in main stem discolored brown to black. Infected trees eventually developed dieback and wilt. Disease incidence ranged from 10% to 22% and 25% to 40% of crown symptoms in the affected coffee trees. In extreme cases, 50% out of 380 trees were affected. The stems of coffee trees with typical symptoms were collected, and then the diseased tissues were surface disinfected with 75% ethanol for 30 s and 0.1% mercuric chloride (HgCl2) solution for 2 min, rinsed three times with sterile distilled water, plated onto potato dextrose agar (PDA) medium, and incubated at 25°C. After 6 days, fungal mycelium was observed growing from the tissue. Three isolates (C3-1, C3-2, and C3-2-1) were obtained by picking spore masses from the apices of perithecia and transferring them to malt extract agar (MEA) medium and incubated at 25°C for 10 days to observe the cultural features. In culture, colonies reaching 65 mm within 10 days, mycelium initially white, then becoming light blue-green. After 6 days of formation, perithecia were black, globose (123.8 - 173.4 μm × 138.2 - 180.6 μm), and showed a long black neck (414.2 - 650.0 μm). Ascospores with outer cell wall forming a brim, hat-shaped, accumulating in a mucilaginous mass at the tips of ostiolar hyphae (4.3 μm × 6.0 μm). Cylindrical endoconidia (14.1 - 45.2 μm × 3.5 - 5.7 μm) were hyaline. Chain of barrel-shaped conidia (6.6 - 10.2 μm × 6.8 - 8.8 μm) were found. Aleuroconidia (10.8 - 16.9 μm × 9.1 - 13.0 μm) were olive-brown, ovoid or obpyriform, and smooth. Morphological characteristics of the fungus were consistent with the description of Ceratocystis fimbriata Ellis & Halst. (Engelbrecht and Harrington 2005). The three isolates were used for molecular identification, and their genomic DNA was extracted using the chelex-100 method (Xu et al. 2020). The internal transcribed spacer (ITS) region of rDNA was sequenced using the procedures of Thorpe et al. (2005). Analysis of the ITS sequence data (GenBank accessions KY580836, KJ511480, and KJ511479) showed that the isolates were 100% homologous to isolates of C. fimbriata from Punica granatum, Camellia sinensis, and Cucumis sativus in China (GenBank accessions KY580891, KY580870, and MH535909, respectively) by BLAST analysis. Neighbor-joining (NJ) phylogenetic analysis was performed using MEGA 6.06 based on the ITS sequences. The three isolates were clustered on the same clade with other C. fimbriata isolates with a high bootstrap value (90%). Therefore, the fungus was identified as C. fimbriata based on both morphological and molecular characteristics. Pathogenicity of the three isolates was tested by inoculating one-year-old pot grown coffee seedlings (C. arabica) through drenching the loams with 30 ml spore suspension (1 × 106 spores/ml). Control plants were inoculated with 30 ml of sterile distilled water. The trees were kept in a controlled greenhouse at 25°C and watered weekly. One month after inoculation, all inoculated plants produced typical dieback and wilt symptoms, whereas the control trees showed no symptoms. The same fungus was isolated from the inoculated trees on PDA and identified as C. fimbriata according to the methods described above, and no fungal growth was observed in the controls, thus fulfilling the Koch's postulates. Coffee canker disease caused by C. fimbriata has been reported in Indonesia and Colombia (Marin et al. 2003). To our knowledge, this is the first report of C. fimbriata causing canker disease of coffee trees in China.

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First Report of Ceratocystis fimbriata-Caused Wilt of Eriobotrya japonica in China

Plant Disease ◽

10.1094/pdis-12-13-1290-pdn ◽

2014 ◽

Vol 98 (9) ◽

pp. 1270-1270 ◽

Cited By ~ 6

Author(s):

J. Li ◽

J. M. Gao ◽

Y. H. Han ◽

Y. X. Sun ◽

Q. Huang

Keyword(s):

Plant Pathogens ◽

Sequence Data ◽

Pcr Amplification ◽

Fungal Growth ◽

Eriobotrya Japonica ◽

Molecular Characteristics ◽

Malt Extract ◽

Ceratocystis Fimbriata ◽

First Report ◽

Initial Symptoms

The loquat (Eriobotrya japonica (Thunb.) Lindl.), an important perennial evergreen fruit crop that originated in China and is also cultivated as an ornamental plant, is well known all over the world for high nutritional, medicinal, economic, and ecological values (4). So far, it has been grown in more than 30 countries (1) and is becoming an important industry in China, Spain, Japan, India, Pakistan, and Turkey (2). During an investigation from May to August of 2013, severely withered loquat plants were observed in Kunming city of Yunnan Province (25°02′ N; 102°42′ E), Southwest China. Initial symptoms were brown lesions of leaves and canker on one to several branches, leaves of the whole tree turned wilted. Cross sections of diseased plants revealed irregularly shaped brown discoloration in the xylem of the trunk. A fungus was consistently isolated from the leaf when diseased leaves were incubated between two slices of fresh carrot root. Spore masses were picked from the apices of perithecia and transferred to malt extract agar medium (MEA) and incubated at 25°C. After perithecium formation, observed perithecia were black, globose (176.0 to 303.2 × 186.0 to 274.3 μm) and showed a long black neck (634.2 to 809.9 μm). Ascospore had the typical format of a “hat” with dimensions of 4.6 to 6.3 × 3.3 to 4.8 μm. Cylindrical endoconidia (7.1 to 36.1 × 2.9 to 6.0 μm) were found. Chlamydospores were ovoid or obpyriform and smooth (8.6 to 12.1 × 6.9 to 12.1 μm). PCR amplification was carried out for one isolate, YT2. The ITS region of rDNA was sequenced using the procedures of Thorpe et al (3). Analysis of ITS sequence data (GenBank Accession Nos. KF963101 and KF963102) showed that the isolates were 99% homologous to the isolate of Ceratocystis fimbriata from diseased Colocasia esculenta in Cuba, China, and Hawaii (AY526304 to 06) by BLAST analysis. Therefore, the fungus was identified as C. fimbriata based on morphological and molecular characteristics. Pathogenicity of the six isolates from this study was tested by inoculation of 1-year-old pot grown seedlings of loquat. The soil of six plants was inoculated by drenching with 40 ml spore suspension (106 spores/ml). Control plants were inoculated with 40 ml of sterile distilled water. The plants were maintained in a controlled greenhouse at 25°C and watered weekly. After inoculation for 1 week, all plants produced wilt symptoms; as the disease progressed, leaves withered and died after 3 weeks of inoculation while control plants remained symptomless. C. fimbriata was successfully re-isolated from the infected trees and no fungal growth was observed in the controls. The pathogenicity assay showed that C. fimbriata was pathogenic to loquat. To our knowledge, this is the first report of C. fimbriata causing wilt of loquat in China. Since C. fimbriata is one of the most aggressive plant pathogens on a wide variety of perennial as well as agronomic crop plants worldwide, and the infection can pose a significant threat to the production of loquat, it is critical to deploy appropriate management strategies to limit the fungus spread. References: (1) J. J. Feng et al. Acta Hort. 750:117, 2007. (2) J. Janick. Acta Hort. 750:27, 2007. (3) D. J. Thorpe et al. Phytopathology 95:316, 2005. (4) J. Yan et al. Pak. J. Bot. 44:1215, 2012.

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First Report of Flyspeck Caused by Zygophiala wisconsinensis on Sweet Persimmon Fruit in Korea

Plant Disease ◽

10.1094/pdis-12-10-0917 ◽

2011 ◽

Vol 95 (5) ◽

pp. 616-616 ◽

Cited By ~ 1

Author(s):

J. Kim ◽

O. Choi ◽

J.-H. Kwon

Keyword(s):

Disease Incidence ◽

Morphological Characteristics ◽

Its Rdna ◽

Control Treatment ◽

Diospyros Kaki ◽

Distilled Water ◽

First Report ◽

Persimmon Fruit ◽

Fungal Isolates ◽

Agar Disc

Sweet persimmon (Diospyros kaki L.), a fruit tree in the Ebenaceae, is cultivated widely in Korea and Japan, the leading producers worldwide (2). Sweet persimmon fruit with flyspeck symptoms were collected from orchards in the Jinju area of Korea in November 2010. The fruit had fungal clusters of black, round to ovoid, sclerotium-like fungal bodies with no visible evidence of a mycelial mat. Orchard inspections revealed that disease incidence ranged from 10 to 20% in the surveyed area (approximately 10 ha) in 2010. Flyspeck symptoms were observed on immature and mature fruit. Sweet persimmon fruit peels with flyspeck symptoms were removed, dried, and individual speck lesions transferred to potato dextrose agar (PDA) and cultured at 22°C in the dark. Fungal isolates were obtained from flyspeck colonies on 10 sweet persimmon fruit harvested from each of three orchards. Fungal isolates that grew from the lesions were identified based on a previous description (1). To confirm identity of the causal fungus, the complete internal transcribed spacer (ITS) rDNA sequence of a representative isolate was amplified and sequenced using primers ITS1 and ITS4 (4). The resulting 552-bp sequence was deposited in GenBank (Accession No. HQ698923). Comparison with ITS rDNA sequences showed 100% similarity with a sequence of Zygophiala wisconsinensis Batzer & Crous (GenBank Accession No. AY598855), which infects apple. To fulfill Koch's postulates, mature, intact sweet persimmon fruit were surface sterilized with 70% ethanol and dried. Three fungal isolates from this study were grown on PDA for 1 month. A colonized agar disc (5 mm in diameter) of each isolate was cut from the advancing margin of a colony with a sterilized cork borer, transferred to a 1.5-ml Eppendorf tube, and ground into a suspension of mycelial fragments and conidia in a blender with 1 ml of sterile, distilled water. The inoculum of each isolate was applied by swabbing a sweet persimmon fruit with the suspension. Three sweet persimmon fruit were inoculated per isolate. Three fruit were inoculated similarly with sterile, distilled water as the control treatment. After 1 month of incubation in a moist chamber at 22°C, the same fungal fruiting symptoms were reproduced as observed in the orchards, and the fungus was reisolated from these symptoms, but not from the control fruit, which were asymptomatic. On the basis of morphological characteristics of the fungal colonies, ITS sequence, and pathogenicity to persimmon fruit, the fungus was identified as Z. wisconsinensis (1). Flyspeck is readily isolated from sweet persimmon fruit in Korea and other sweet persimmon growing regions (3). The exposure of fruit to unusual weather conditions in Korea in recent years, including drought, and low-temperature and low-light situations in late spring, which are favorable for flyspeck, might be associated with an increase in occurrence of flyspeck on sweet persimmon fruit in Korea. To our knowledge, this is the first report of Z. wisconsinensis causing flyspeck on sweet persimmon in Korea. References: (1) J. C. Batzer et al. Mycologia 100:246, 2008. (2) FAOSTAT Database. Retrieved from http://faostat.fao.org/ , 2008. (3) H. Nasu and H. Kunoh. Plant Dis. 71:361, 1987. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, Inc., New York, 1990.

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CYTOTOXIC BIOACTIVE COMPOUNDS FROM CALOTROPIS GIGANTEA STEM BARK

International Journal of Pharmacy and Pharmaceutical Sciences ◽

10.22159/ijpps.2016.v8i9.12430 ◽

2016 ◽

Vol 8 (9) ◽

pp. 111

Author(s):

Kartini Hasballah ◽

Murniana . ◽

Erya . ◽

Ardian .

Keyword(s):

Ethyl Acetate ◽

Cytotoxic Activity ◽

Ethyl Acetate Extract ◽

Leukemia Cell ◽

Stem Bark ◽

Hexane Extract ◽

Leukemia Cell Line ◽

Calotropis Gigantea ◽

Murine Leukemia Cell ◽

Main Components

Objective: The present study deals with the cytotoxic activity of n-hexane and ethyl acetate extracts of Calotropis gigantea L. stem bark and its fractions such as A, B, C, D and E fractions on murine leukemia cell line P388.Methods: The crude extracts of C. gigantea stem bark were prepared using n-hexane and ethyl acetate solvents. The plant extracts were subjected to vacuum liquid chromatography followed by TLC. According to the similarity of stain patterns, the fractions were combined. The extracts and its combined fractions were then subjected for the phytochemical test. Cytotoxic activity of those extracts and its combined fractions were tested using MTT assay. Fraction D was subjected to gravity column chromatography followed by TLC. Then, fractions A, B, and D2 were crystallized and subjected to GC-MS.Results: The qualitative screening of n-hexane extract of Calotropis gigantea L. stem bark for secondary metabolites showed the presence of terpenoid, flavonoids, phenolics and coumarins. While the ethyl acetate extract contained phenolics, steroids, flavonoids, saponins and coumarins compounds. IC50 values for n-hexane extract and E fraction are 76.29 µg/ml and 18.48 µg/ml, respectively. In the ethyl acetate extract and C fraction obtained IC50 values 57.05 µg/ml and 52.58 µg/ml.Conclusion: Cytotoxic activity from E fraction of n-hexane extract of C. gigantea stem bark is the most potent and containing flavonoids, phenolics and coumarins. The main components from several compounds of n-hexane extract of C. gigantea are germacrane-A, (-)-globulol, urs-12-ene and veridiflorol.

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First Report of Alternaria Leaf Spot Caused by Alternaria sp. on Highbush Blueberry (Vaccinium corymbosum) in South Korea

Plant Disease ◽

10.1094/pdis-04-14-0344-pdn ◽

2014 ◽

Vol 98 (10) ◽

pp. 1434-1434

Author(s):

J.-H. Kwon ◽

D.-W. Kang ◽

M.-G. Cheon ◽

J. Kim

Keyword(s):

South Korea ◽

Leaf Spot ◽

Disease Incidence ◽

Vaccinium Corymbosum ◽

Its Rdna ◽

Universal Primers ◽

Conidial Suspension ◽

First Report ◽

Representative Isolate ◽

Alternaria Sp

In South Korea, the culture, production, and consumption of blueberry (Vaccinium corymbosum) have increased rapidly over the past 10 years. In June and July 2012, blueberry plants with leaf spots (~10% of disease incidence) were sampled from a blueberry orchard in Jinju, South Korea. Leaf symptoms included small (1 to 5 mm in diameter) brown spots that were circular to irregular in shape. The spots expanded and fused into irregularly shaped, large lesions with distinct dark, brownish-red borders. The leaves with severe infection dropped early. A fungus was recovered consistently from sections of surface-disinfested (1% NaOCl) symptomatic leaf tissue after transfer onto water agar and sub-culture on PDA at 25°C. Fungal colonies were dark olive and produced loose, aerial hyphae on the culture surfaces. Conidia, which had 3 to 6 transverse septa, 1 to 2 longitudinal septa, and sometimes also a few oblique septa, were pale brown to golden brown, ellipsoid to ovoid, obclavate to obpyriform, and 16 to 42 × 7 to 16 μm (n = 50). Conidiophores were pale to mid-brown, solitary or fasciculate, and 28 to 116 × 3 to 5 μm (n = 50). The species was placed in the Alternaria alternata group (1). To confirm the identity of the fungus, the complete internal transcribed spacer (ITS) rDNA region of a representative isolate, AAVC-01, was amplified using ITS1 and ITS4 primers (2). The DNA products were cloned into the pGEM-T Easy vector (Promega, Madison, WI) and the resulting pOR13 plasmid was sequenced using universal primers. The resulting 570-bp sequence was deposited in GenBank (Accession No. KJ636460). Comparison of ITS rDNA sequences with other Alternaria spp. using ClustalX showed ≥99% similarity with the sequences of A. alternata causing blight on Jatropha curcas (JQ660842) from Mexico and Cajannus cajan (JQ074093) from India, citrus black rot (AF404664) from South Africa, and other Alternaria species, including A. tenuissima (WAC13639) (3), A. lini (Y17071), and A. longipes (AF267137). Two base substitutions, C to T at positions 345 and 426, were found in the 570-bp amplicon. Phylogenetic analysis revealed that the present Alternaria sp. infecting blueberry grouped separately from A. tenuissima and A. alternata reported from other hosts. A representative isolate of the pathogen was used to inoculate V. corymbosum Northland leaves for pathogenicity testing. A conidial suspension (2 × 104 conidia/ml) from a single spore culture and 0.025% Tween was spot inoculated onto 30 leaves, ranging from recently emerged to oldest, of 2-year-old V. corymbosum Northland plants. Ten leaves were treated with sterilized distilled water and 0.025% Tween as a control. The plants were kept in a moist chamber with >90% relative humidity at 25°C for 48 h and then moved to a greenhouse. After 15 days, leaf spot symptoms similar to those observed in the field developed on the inoculated leaves, whereas the control plants remained asymptomatic. The causal fungus was re-isolated from the lesions of the inoculated plants to fulfill Koch's postulates. To our knowledge, this is the first report of Alternaria sp. on V. corymbosum in South Korea. References: (1) E. G. Simmons. Page 1797 in: Alternaria: An Identification Manual. CBS Fungal Biodiversity Centre, 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, 1990. (3) M. P. You et al. Plant Dis. 98:423, 2014.

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First report of Anthracnose on Peach Fruit Caused by Colletotrichum siamense in Uruguay

Plant Disease ◽

10.1094/pdis-05-21-0950-pdn ◽

2021 ◽

Author(s):

María Julia Carbone ◽

Victoria Moreira ◽

Pedro Mondino ◽

Sandra Alaniz

Keyword(s):

South Carolina ◽

Prunus Persica ◽

Disease Incidence ◽

Management Strategies ◽

Control Treatment ◽

Fluorescent Light ◽

Peach Fruit ◽

First Report ◽

Anthracnose Disease ◽

Equidistant Points

Peach (Prunus persica L.) is an economically important deciduous fruit crop in Uruguay. Anthracnose caused by species of the genus Colletotrichum is one of the major diseases in peach production, originating significant yield losses in United States (Hu et al. 2015), China (Du et al. 2017), Korea (Lee et al. 2018) and Brazil (Moreira et al. 2020). In February 2017, mature peach fruits cv. Pavia Canario with symptoms resembling anthracnose disease were collected from a commercial orchard located in Rincon del Colorado, Canelones, in the Southern region of Uruguay. Symptoms on peach fruit surface were characterized as circular, sunken, brown to dark-brown lesions ranging from 1 to 5 cm in diameter. Lesions were firm to touch with wrinkled concentric rings. All lesions progressed to the fruit core in a V-shaped pattern. The centers of the lesions were covered by orange conidial masses. Monosporic isolates obtained from the advancing margin of anthracnose lesions were grown on PDA at 25ºC and 12h photoperiod under fluorescent light. The representative isolates DzC1, DzC2 and DzC6 were morphologically and molecularly characterized. Upper surface of colonies varied from white or pale-gray to gray and on the reverse dark-gray with white to pale-gray margins. Conidia were cylindrical, with both ends predominantly rounded or one slightly acute, hyaline and aseptate. The length and width of conidia ranged from 9.5 to 18.9 µm (x ̅=14.1) and from 3.8 to 5.8 µm (x ̅=4.6), respectively. The ACT, βTUB2, GAPDH, APN2, APN2/MAT-IGS, and GAP2-IGS gene regions were amplified and sequenced with primers ACT-512F/ACT-783R (Carbone and Kohn, 1999), BT2Fd/BT4R (Woudenberg et al. 2009), GDF1/GDR1 (Guerber et al. 2003), CgDLR1/ColDLF3, CgDLF6/CgMAT1F2 (Rojas et al. 2010) and GAP1041/GAP-IGS2044 (Vieira et al. 2017) respectively and deposited in the GenBank database (MZ097888 to MZ097905). Multilocus phylogenetic analysis revealed that Uruguayan isolates clustered in a separate and well supported clade with sequences of the ex-type (isolate ICMP 18578) and other C. siamense strains (isolates Coll6, 1092, LF139 and CMM 4248). To confirm pathogenicity, mature and apparently healthy peach fruit cv. Pavia Canario were inoculated with the three representative isolates of C. siamense (six fruit per isolate). Fruit were surface disinfested with 70% ethanol and wounded with a sterile needle at two equidistant points (1 mm diameter x 1 mm deep). Then, fruit were inoculated with 5 µl of a spore suspension (1×106 conidia mL-1) in four inoculation points per fruit (two wounded and two unwounded). Six fruit mock-inoculated with 5 µl sterile water were used as controls. Inoculated fruit were placed in moist chamber and incubated at 25°C during 10 days. Anthracnose lesions appeared at 2 and 4 days after inoculation in wounded and unwounded points, respectively. After 7 days, disease incidence was 100% and 67% for wounded and unwounded fruit, respectively. The control treatment remained symptomless. The pathogens were re-isolated from all lesions and re-identified as C. siamense. C. siamense was previously reported in South Carolina causing anthracnose on peach (Hu et al. 2015). To our knowledge, this is the first report of anthracnose disease on peach caused by C. siamense in Uruguay. Effective management strategies should be implemented to control anthracnose and prevent the spread of this disease to other commercial peach orchards.

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