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

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

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First Report of Lasiodiplodia theobromae Causing Inflorescence Blight of Mango

Plant Disease ◽

10.1094/pdis-03-13-0238-pdn ◽

2013 ◽

Vol 97 (10) ◽

pp. 1380-1380 ◽

Cited By ~ 2

Author(s):

L. M. Serrato-Diaz ◽

M. Perez-Cuevas ◽

L. I. Rivera-Vargas ◽

R. D. French-Monar

Keyword(s):

Puerto Rico ◽

Mangifera Indica ◽

Fruit Rot ◽

Its2 Region ◽

Morphological Identification ◽

Tropical Fruit ◽

Lasiodiplodia Theobromae ◽

First Report ◽

Pure Cultures ◽

Β Tubulin

Mango (Mangifera indica L.) is an important tropical fruit crop in Puerto Rico. During a disease survey from 2008 to 2010, inflorescence blight was observed at the Mango Germplasm Collection of the University of Puerto Rico's Experiment Station in Juana Diaz as a rotting of the rachis (main axis of the inflorescence), rachilla (lateral axis), and flowers. Diseased inflorescences from cultivars ‘Haden’ and ‘Irwin’ were disinfested with 70% ethanol, followed by 0.5% sodium hypochlorite, rinsed with sterile water, and transferred to acidified potato dextrose agar (APDA). Two isolates of Lasiodiplodia theobromae (Pat.) Griffon & Maubl. were isolated from symptomatic tissue and identified morphologically using a Botryosphaeriaceae taxonomic key (3). In APDA, colonies of L. theobromae had initial greenish gray aerial mycelia that turned dark brown with age. Pycnidia were uniloculate and dark brown to black in color. Conidiogenous cells were hyaline, cylindrical, and holoblastic. Immature conidia were subovoid to ellipsoid, apex rounded, truncate at the base, thick walled, hyaline and one-celled, becoming dark brown, two-celled with irregular longitudinal striations when mature. Conidia (n = 50) averaged 26.88 μm long by 12.98 μm wide. Genomic DNA was extracted from pure cultures using a Qiagen DNeasy Plant Mini Kit. PCR amplification of three genes was used to support morphological identification. DNA analysis of the ITS1-5.8S-ITS2 region, and fragments of both β-tubulin and elongation factor 1 alpha (EF1-α) genes were sequenced and compared using BLASTN with sequences available in GenBank. Accession numbers of gene sequences of L. theobromae from Puerto Rico submitted to GenBank were: KC631659 and KC631660 for ITS region; KC631651 and KC631652 for β-tubulin; and KC631655 and KC631656 for EF1α. For all genes used, sequences were 99 to 100% identical to reference isolate CBS164.96 of L. theobromae reported in GenBank. Pathogenicity tests were conducted on six random healthy non-detached mango inflorescences from cultivars Haden and Irwin. Inflorescences were inoculated with 5-mm mycelial disks from 8-day-old pure cultures grown in APDA and kept in a humid chamber using plastic bags for 8 days under field conditions. Untreated controls were inoculated with APDA disks only. The test was repeated twice. For both cultivars, isolates of L. theobromae caused inflorescence (rachis, rachilla, and flowers) blight, 8 days after inoculation. Inflorescences turned brown and profuse mycelial growth was observed on the inflorescences. Untreated controls were disease-free and no fungi were reisolated from tissue. L. theobromae was reisolated from diseased inflorescences, fulfilling Koch's postulates. Fungi in the family Botryosphaeriaceae have been associated with stem-end rot, fruit rot, branch dieback, blossom blight, and cankers on mango (1,2,4). Worldwide, L. theobromae has only been reported causing dieback, stem end rot and fruit rot in mango (1,2). To our knowledge, this is the first report of L. theobromae causing inflorescence blight in mango. References: (1) N. I. Hui-Fang et al. Botanical Stud. 53:467, 2012. (2) A. M. Ismail et al. Australas. Plant Pathol. 41:649, 2012. (3) A. J. L. Phillips. Key to the various lineages in “Botryosphaeria” Version 01 2007. Retrieved from http://www.crem.fct.unl.pt/botryosphaeria_site/key.htm , 6 August 2013. (4) B. Slippers et al. Mycologia 97:99, 2005.

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First Report of Diaporthe pseudomangiferae Causing Inflorescence Rot, Rachis Canker, and Flower Abortion of Mango

Plant Disease ◽

10.1094/pdis-12-13-1223-pdn ◽

2014 ◽

Vol 98 (7) ◽

pp. 1004-1004 ◽

Cited By ~ 1

Author(s):

L. M. Serrato-Diaz ◽

L. I. Rivera-Vargas ◽

R. D. French-Monar

Keyword(s):

Dna Analysis ◽

Mangifera Indica ◽

Germplasm Collection ◽

Its2 Region ◽

Post Inoculation ◽

Tropical Fruit ◽

First Report ◽

Flower Abortion ◽

Wide Range ◽

Β Tubulin

Although mango (Mangifera indica L.) is a very important tropical fruit crop, limited studies have been conducted on fungal pathogens affecting the inflorescences. During a disease survey conducted from 2008 to 2010, 50% of the inflorescences were affected with inflorescence rot, rachis canker, and flower abortion characterized by blackening of plant tissue with soft rot lesions and suken lesions on the rachis, respectively. Symptoms were observed at the Mango Germplasm Collection of the University of Puerto Rico's Experiment Station in Juana Diaz, Puerto Rico. Five diseased pieces of 350 inflorescences from cvs. Haden and Irwin were disinfested with 70% ethanol, followed by 0.5% sodium hypochlorite, rinsed with sterile water, and transferred to acidified potato dextrose agar (APDA). Among several typical or common fungi, three isolates of Diaporthe pseudomangiferae (Dp) R.R. Gomes, C. Glienke & Crous were obtained from symptomatic tissue and identified morphologically using taxonomic keys and DNA sequence comparisons (1,2). On APDA, colonies of Dp initially had white-gray moderate aerial mycelia. Pycnidia were black and superficial on cultures with a central ostiole that exuded beige to light orange conidial droplets. Alpha conidia (n = 50) were aseptate, hyaline, smooth, fusiform, apex rounded and base truncate, averaged 7.34 μm long by 2.60 μm wide. Beta conidia (n = 50) were spindle-shaped, aseptate, hyaline and smooth, averaged 22.03 μm long by 1.53 μm wide. DNA analysis of the ITS1-5.8S-ITS2 region using primers ITS5 and ITS4, and fragments of both β-tubulin and translation elongation factor 1 alpha (EF1-α) genes using primers T1 and Bt2b, and EF1-728F and EF1-986R, respectively, were sequenced and compared using BLASTn with sequences available in the GenBank. Accession numbers of gene sequences of Dp submitted to GenBank were KF616498 to KF616500 for ITS region, KF616501 to KF616503 for β-tubulin, and KF616504 to KF616506 for EF1-α. For all genes used, sequences were 99 to 100% identical to reference isolate CBS 388.89 of Dp in GenBank. For each fungal isolate, pathogenicity tests were conducted on six random healthy non-detached mango inflorescences for both cvs. Haden and Irwin. Inflorescences were inoculated with 5-mm mycelial disks from 8-day-old pure cultures grown on APDA and kept in a humid chamber using plastic bags for 8 days under field conditions. Untreated controls were inoculated with APDA disks only. The test was repeated twice. On cv. Haden, isolates of Dp caused rachis canker (sunken lesion on the rachis) at 8 days post inoculation (dpi). On cv. Irwin, isolates of Dp caused inflorescence rot. Initially, white mycelia was observed on inflorescences but eventually inflorescences turned brown and flower abortion was observed at 8 dpi. Untreated controls did not show any of the above symptoms and no fungi were re-isolated from tissue. From diseased inflorescences, Dp was re-isolated, thus fulfilling Koch's postulates. Diaporthe spp. have been associated with fruit rots, stem cankers, decay, and wilt on a wide range of plant hosts (3,4). Recently, Dp was associated with fruit peel of mango in Mexico and the Dominican Republic (2). To our knowledge, this is the first report of Dp causing inflorescence rot, rachis canker, and flower abortion in mango. References: (1) H. L. Barnett and B. B. Hunter. Illustrated Genera of Imperfect Fungi. APS Press. St. Paul, MN, 1998. (2) R. R. Gomes et al. Persoonia. 31:1, 2013. (3) J. M. Santos et al. Persoonia 27:9, 2011. (4) S. M. Thompson et al. Persoonia 27:80, 2011.

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First record of Colletotrichum alienum Causing postharvest Anthracnose disease of mango fruit in China

Plant Disease ◽

10.1094/pdis-09-20-2074-pdn ◽

2021 ◽

Author(s):

Tanvir Ahmad ◽

Jingjing Wang ◽

Yongquan Zheng ◽

Ankwasa Edgar Mugizi ◽

Anam Moosa ◽

...

Keyword(s):

Fruit Rot ◽

Post Inoculation ◽

Pathogenicity Test ◽

Distilled Water ◽

Mango Fruit ◽

Causal Organism ◽

Isolation Technique ◽

Anthracnose Disease ◽

Colletotrichum Species ◽

Fruit Surface

Mango (Mangifera indica L.) is one of the world's most significant economic fruit crops, and China is the second-largest producer of mango (Kuhn et al., 2017). Postharvest mango anthracnose is caused by Colletotrichum species and reduce the self-life of mature fruit (Wu et al., 2020). Colletotrichum species also cause postharvest anthracnose and fruit rot disease of Apple, Banana and Avocado (Khodadadi et al., 2020; Vieira et al., 2017; Sharma et al., 2017). In July 2019, mango fruits cv. ‘Jin-Hwang’ were observed at different fruit markets (39°48'42.1"N 116°20'17.0"E) of the Fengtai district, Beijing, China, exhibiting typical symptoms of anthracnose including brown to black lesions in different size (≤ 2 cm) with identified border on the mango fruit surface. Later, the lesions were coalesced and extensively cover the surface area of the fruit. The lesions were also restricted to peel the fruit and pathogen invaded in the fruit pulp. About 30% of mango fruits were affected by anthracnose disease. The margins of lesions from infected mango fruits (n=56) were cut into 2 × 2 mm pieces, surface disinfected with NaClO (2% v/v) for 30 s, rinsed thrice with distilled water for 60s. These pieces were placed on PDA medium and incubated at 25°C for 7 days. Pure culture of fungal isolates was obtained by single spore isolation technique. Initially, the fungal colony was off white, and colony extended with time, turning light gray at the center. The morphological examination revealed that conidia were hyaline, oblong, and unicellular. The conidia were measured from 10 days old culture and dimensions varied from 13.3 to 15.8 µm in length and 4.6 to 6.1 µm in width. For molecular identification, a multi-locus sequence analysis; the Internal Transcribed Spacers (ITS) region, partial actin (ACT) gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene and chitin synthase (CHS-1) gene were amplified by using the primer sets ITS1/4 (White et al. 1990), ACT-512F/ACT-783R (Carbone and Kohn 1999), GDF1/GDR1 (Guerber et al. 2003) and CHS1-79F/CHS-1-354R (Carbone and Kohn 1999) respectively. The partial sequences of MTY21 were deposited to GenBank accessions (MT921666 (ITS), MT936119 (ACT), MT936120 (GAPDH) and MT936118 (CHS-1). All obtained sequences showed 100% similarity with reported sequences of Colletotrichum alienum ICMP.18691 with accessions numbers JX010217 (ITS), JX009580 (ACT), JX010018 (GAPDH) and JX009754 (CHS-1) which represented the isolate MTY21 identified as C. alienum by constructing Maximum Likelihood phylogenetic tree analysis using Mega X (Kumar et al., 2018). For the confirmation of Koch's postulates, the pathogenicity test was conducted on 36 fresh healthy mango fruits for each treatment. Fruits were punctured with the help of a sterilized needle to create 2mm2 wounds and inoculated with 10µL inoculum (107 spores/mL) of MTY21. Control mango fruits were inoculated with 10µL sterilized distilled water and incubated at 25 °C with 90% relative humidity. The lesions appeared at the point of inoculation and gradually spread on the fruit surface after 7 days post inoculation. The symptoms were similar to the symptoms on original fruit specimens. The re-isolated fungus was identified as C. alienum based on morphological and molecular analysis. Mango anthracnose disease caused by several Colletotrichum species has been reported previously on mango in China (Li et al., 2019). Liu et al. (2020) reported C. alienum as the causal organism of anthracnose disease on Aquilaria sinensis in China. C. alienum has been previously reported causing mango anthracnose disease in Mexico (Tovar-Pedraza et al., 2020) To our knowledge, this is the first report of C. alienum causing postharvest anthracnose of mango in China. The prevalence of C. alienum was 30% on mango fruit which reflects the importance of this pathogen as a potential problem of mango fruit in China.

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First Report of Pepper Fruit Rot Caused by Fusarium concentricum in China

Plant Disease ◽

10.1094/pdis-03-13-0325-pdn ◽

2013 ◽

Vol 97 (12) ◽

pp. 1657-1657 ◽

Cited By ~ 6

Author(s):

J. H. Wang ◽

Z. H. Feng ◽

Z. Han ◽

S. Q. Song ◽

S. H. Lin ◽

...

Keyword(s):

Fusarium Species ◽

Fruit Rot ◽

Post Inoculation ◽

Distilled Water ◽

Conidial Suspension ◽

Average Growth ◽

First Report ◽

Control Fruit ◽

Pepper Fruit ◽

Fruit Samples

Pepper (Capsicum annuum L.) is an important vegetable crop worldwide. Some Fusarium species can cause pepper fruit rot, leading to significant yield losses of pepper production and, for some Fusarium species, potential risk of mycotoxin contamination. A total of 106 diseased pepper fruit samples were collected from various pepper cultivars from seven provinces (Gansu, Hainan, Heilongjiang, Hunan, Shandong, Shanghai, and Zhejiang) in China during the 2012 growing season, where pepper production occurs on approximately 25,000 ha. Pepper fruit rot symptom incidence ranged from 5 to 20% in individual fields. Symptomatic fruit tissue was surface-sterilized in 0.1% HgCl2 for 1 min, dipped in 70% ethanol for 30 s, then rinsed in sterilized distilled water three times, dried, and plated in 90 mm diameter petri dishes containing potato dextrose agar (PDA). After incubation for 5 days at 28°C in the dark, putative Fusarium colonies were purified by single-sporing. Forty-three Fusarium strains were isolated and identified to species as described previously (1,2). Morphological characteristics of one strain were identical to those of F. concentricum. Aerial mycelium was reddish-white with an average growth rate of 4.2 to 4.3 mm/day at 25°C in the dark on PDA. Pigments in the agar were formed in alternating red and orange concentric rings. Microconidia were 0- to 1-septate, mostly 0-septate, and oval, obovoid to allantoid. Macroconidia were relatively slender with no significant curvature, 3- to 5-septate, with a beaked apical cell and a foot-shaped basal cell. To confirm the species identity, the partial TEF gene sequence (646 bp) was amplified and sequenced (GenBank Accession No. KC816735). A BLASTn search with TEF gene sequences in NCBI and the Fusarium ID databases revealed 99.7 and 100% sequence identity, respectively, to known TEF sequences of F. concentricum. Thus, both morphological and molecular criteria supported identification of the strain as F. concentricum. This strain was deposited as Accession MUCL 54697 (http://bccm.belspo.be/about/mucl.php). Pathogenicity of the strain was confirmed by inoculating 10 wounded, mature pepper fruits that had been harvested 70 days after planting the cultivar Zhongjiao-5 with a conidial suspension (1 × 106 spores/ml), as described previously (3). A control treatment consisted of inoculating 10 pepper fruits of the same cultivar with sterilized distilled water. The fruit were incubated at 25°C in a moist chamber, and the experiment was repeated independently in triplicate. Initially, green to dark brown lesions were observed on the outer surface of inoculated fruit. Typical soft-rot symptoms and lesions were observed on the inner wall when the fruit were cut open 10 days post-inoculation. Some infected seeds in the fruits were grayish-black and covered by mycelium, similar to the original fruit symptoms observed at the sampling sites. The control fruit remained healthy after 10 days of incubation. The same fungus was isolated from the inoculated infected fruit using the method described above, but no fungal growth was observed from the control fruit. To our knowledge, this is the first report of F. concentricum causing a pepper fruit rot. References: (1) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA, 2006. (2) K. O'Donnell et al. Proc. Nat. Acad. Sci. USA 95:2044, 1998. (3) Y. Yang et al. 2011. Int. J. Food Microbiol. 151:150, 2011.

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

Plant Disease ◽

10.1094/pdis-10-20-2173-pdn ◽

2021 ◽

Author(s):

Yuexuan Long ◽

Mingxue Shang ◽

Yue Deng ◽

Chuan Yu ◽

Mingde Wu ◽

...

Keyword(s):

Brassica Juncea ◽

Yellow Pigment ◽

Causal Agent ◽

Control Group ◽

Conidial Suspension ◽

First Report ◽

Fungal Isolates ◽

Dna Fragment ◽

Leptosphaeria Biglobosa ◽

Β Tubulin

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

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First Report of Lasiodiplodia theobromae Causing Inflorescence Blight and Fruit Rot of Longan (Dimocarpus longan L.) in Puerto Rico

Plant Disease ◽

10.1094/pdis-05-13-0473-pdn ◽

2014 ◽

Vol 98 (2) ◽

pp. 279-279 ◽

Cited By ~ 4

Author(s):

L. M. Serrato-Diaz ◽

L. I. Rivera-Vargas ◽

R. Goenaga ◽

R. D. French-Monar

Keyword(s):

Puerto Rico ◽

Fruit Rot ◽

Fluorescent Light ◽

Its2 Region ◽

Tropical Fruit ◽

Lasiodiplodia Theobromae ◽

First Report ◽

Wide Range ◽

Dimocarpus Longan ◽

Β Tubulin

Dimocarpus longan L., commonly known as longan, is a tropical fruit tree of the Sapindaceae family. From 2008 to 2010, a disease survey for longan was conducted in March and October in Puerto Rico. Fruit rot and inflorescence blight (rotting of the rachis, rachilla, and flowers) were observed in fields of longan at the USDA-ARS Research Farm in Isabela, and two commercial orchards in Puerto Rico. Tissue sections (1 mm2) of diseased inflorescences and surface of the fruit were disinfested with 70% ethanol, rinsed with sterile water, and transferred to acidified potato dextrose agar (APDA). Three isolates of Lasiodiplodia theobromae (Pat.) Griffon & Maubl. (Lt) were isolated from symptomatic tissue and identified morpho-molecularly using a taxonomic key for the Botryosphaeriaceae and DNA sequence analysis (1). In APDA, colonies of Lt had initial greenish-gray aerial mycelia that turned dark brown with age. Pycnidia were dark brown to black. Immature conidia were sub-ovoid to ellipsoid, apex rounded, truncate at the base, thick-walled, hyaline, and one-celled, becoming dark brown, two-celled, and with irregular longitudinal striations when mature. Conidia (n = 50) for all the isolates averaged 26.9 μm long by 13 μm wide. For molecular identification, the ITS1-5.8S-ITS2 region and fragments of the β-tubulin and elongation factor 1-alpha (EF1-α) genes were sequenced and BLASTn searches done in GenBank. Accession numbers of gene sequences of Lt submitted to GenBank were KC964546, KC964547, and KC964548 for ITS region; KC964549, KC964550, and KC964551 for β-tubulin; and KC964552, KC964553, and KC964554 for EF1-α. For all genes used, sequences were 99 to 100% identical to reference isolate CBS164.96 of Lt reported in GenBank (accessions AY640255, EU673110, and AY640258). Pathogenicity tests were conducted on six random healthy non-detached inflorescences of longan and six healthy detached fruits per isolate. Unwounded inflorescences and fruit were inoculated with 5-mm mycelial disks from 8-day-old pure cultures grown in APDA. Inflorescences were enclosed in plastic bags for 5 days under field conditions while fruits were kept in a humid chamber using plastic boxes for 5 days under laboratory conditions of 25°C and 12 h of fluorescent light. Untreated controls were inoculated with APDA disks only. The experiment was repeated once. Five days after inoculation, isolates of Lt caused inflorescence blight, fruit rot, and aril (flesh) rot. Inflorescences turned brown and flower mummification was observed on the inflorescences. The exocarp (peel) and endocarp (aril) turned dark brown and mycelial growth and pycnidia of Lt were observed on fruits. Untreated controls did not show any symptoms and no fungi were re-isolated from tissue. In diseased inflorescences and fruits, Lt was re-isolated from diseased tissue and identified using morphological and molecular parameters, thus fulfilling Koch's postulates. Lt has been reported to cause dieback, stem end rot, and fruit rot on a wide range of plants host (2,4). In longan, Lt has been reported causing fruit rot in Thailand (3). To our knowledge, this is the first time that Lt has been reported causing inflorescence blight in longan and the first report of Lt causing fruit rot in Puerto Rico. References: (1) A. J. L. Phillips. Key to the various lineages in “Botryosphaeria” Version 01 2007. Retrieved from http://www.crem.fct.unl.pt/botryosphaeria_site/key.htm , 26 November 2013. (2) B. Slippers et al. Mycologia 97:99, 2005. (3) P. Suwanakood et al. Asian J. Biol. Ed. 3:47, 2007. (4) A. F. Wright and P. F. Harmon. Plant Dis. 93:962, 2009.

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First Report of Internal Black Rot Caused byNeoscytalidium dimidiatumonHylocereus undatus(Pitahaya) Fruit in Israel

Plant Disease ◽

10.1094/pdis-05-13-0535-pdn ◽

2013 ◽

Vol 97 (11) ◽

pp. 1513-1513 ◽

Cited By ~ 9

Author(s):

D. Ezra ◽

O. Liarzi ◽

T. Gat ◽

M. Hershcovich ◽

M. Dudai

Keyword(s):

Its Region ◽

Stem Canker ◽

Brown Spot ◽

Black Rot ◽

Post Inoculation ◽

Single Spore ◽

Flower Opening ◽

Disease Symptoms ◽

First Report ◽

Β Tubulin

Pitahaya (Hylocereus undatus [Haw.] Britton & Rose) was introduced to Israel in 1994, and is grown throughout the country. In the summer of 2009, fruit with internal black rot was collected from a field in central Israel. Symptomatic tissue from the black rot was placed on potato dextrose agar (PDA) plates amended with 12 μg/ml tetracycline and incubated at 25°C for 3 days. A dark, gray to black, fast-growing fungus was isolated from all samples (10 fruits). For identification, single-spore cultures were grown on PDA at 25°C for 5 days, and colonies with gray to black, wooly mycelium were formed. The mycelia were branched and septate (4 to 8 μm wide). The arthroconidia were dark brown, thick-walled, and one-celled, 6.3 to 14.2 × 2.0 to 4.5 μm (n = 5), and ovate to rectangular. Based on these characteristics, the fungus was identified as Neoscytalidium dimidiatum (Penz.) Crous & Slippers (1). The internal transcribed spacer (ITS) region of rDNA and β-tubulin gene were amplified using ITS1 and ITS4, T121 (2), and Bt1b (3) primers, respectively, and then sequenced (GenBank Accessions KF000372 and KF020895, respectively). Both sequences were identical to sequences previously deposited in GenBank. The ITS (561 bp) and β-tubulin (488 bp) sequences exhibited 99% and 100% identity, and 100% and 84% coverage, respectively, to N. dimidiatum (JX524168 and FM211185, respectively). Thus, the results of the molecular identifications confirmed the morphological characterization. To establish fungal pathogenicity and the mechanism of infection, 60 flowers in a disease-free orchard were marked to form three different treatments (15 flowers per treatment): inoculations of the flower tube by inserting PDA plugs (0.5 × 0.5 cm) from a 5-day-old culture to the base of the flower, inoculations of the flower stigma by placing the fungus plug on intact, or pre-wounded flower stigma. The wounds were made by scratching the stigma with a sterile scalpel. For each treatment, five additional flowers were used as negative controls in which the PDA plugs did not contain any fungus. All flowers were hand-pollinated and left to grow for a month until the fruit had ripened. Only flowers inoculated by insertion of the fungus into the flower tube developed black rot in the fruit (8 of 15 fruit) 3 to 4 weeks post inoculation, suggesting involvement of the flower tube in the mechanism of infection. All other treatments and controls failed to develop any detectable disease symptoms. N. dimidiatum was reisolated from the rot, fulfilling Koch's postulates. Flowers with wounded stigma developed significantly smaller fruit. Interestingly, diseased fruit changed color about a week before ripening from the flower opening downwards, whereas healthy fruit changed color from the attachment point to the stem upwards. These results indicate that N. dimidiatum is the pathogen of pitahaya internal black rot disease. Recently, this pathogen was reported to cause brown spot disease and stem canker disease of pitahaya in China (4) and Taiwan (5), respectively. To date, the disease can be detected in all orchards in Israel, with up to 50% of the fruit being infected. Since the disease symptoms of the Israeli isolate are located in the fruit, the commercial loss due to pathogen attack is significant. To our knowledge, this is the first report of internal black rot caused by N. dimidiatum on pitahaya fruit in Israel.References: (1) P. W. Crous et al. Stud. Mycol. 55:235, 2006. (2) K. O'Donnell and E. Cigelnik. Mol. Phylo, Evol. 7:103, 1997. (3) N. L. Glass and G. C. Donaldson. Appl. Environ. Microiol. 61:1323, 1995. (4) G. B. Lan and Z. F. He. Plant Dis. 96:1702, 2012. (5) M. F. Chuang et al. Plant Dis. 96:906, 2012.

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First Report of Neofusicoccum mangiferae Causing Rachis Necrosis and Inflorescence Blight of Mango (Mangifera indica) in Puerto Rico

Plant Disease ◽

10.1094/pdis-08-13-0878-pdn ◽

2014 ◽

Vol 98 (4) ◽

pp. 570-570 ◽

Cited By ~ 2

Author(s):

L. M. Serrato-Diaz ◽

L. I. Rivera-Vargas ◽

R. D. French-Monar

Keyword(s):

Puerto Rico ◽

Mycelial Growth ◽

Mangifera Indica ◽

Elongation Factor ◽

Germplasm Collection ◽

Its Region ◽

Internal Transcribed Spacer Region ◽

First Report ◽

Reference Isolate ◽

Β Tubulin

Inflorescence blight is a major disease in mango production (2,3). During a disease survey of mango in Puerto Rico conducted from February to April in 2009, 20% of the inflorescences were affected with inflorescence blight showing rachis and flower necrosis. Symptoms were observed in 70% of samples at the Mango Germplasm Collection of the University of Puerto Rico's Experiment Station in Juana Diaz. Blighted inflorescence tissue (necrotic and the interface between necrotic and healthy tissue) from mango cultivars ‘Haden’ and ‘Irwin’ were disinfested with 70% ethanol, rinsed with sterile water and transferred to acidified potato dextrose agar (APDA). Isolations (40%) produced fungi in the Botryosphaeriaceae. Isolates 90LY, 94LY, and 89LY were purified and identified morphologically using taxonomic keys (1,4) and by DNA sequence analyses as Neofusicoccum mangiferae (Syd. & P. Syd.) Crous, Slippers & A.J.L. Phillips. On APDA, colonies were gray with aerial mycelia that turned dark gray with age. Pycnidia were globose to pyriform and dark brown to black. Conidia (n = 50) were hyaline, ovoid, one-celled, and averaged 13.2 × 6.3 μm in size. PCR amplifications of the internal transcribed spacer region of rDNA using ITS5-ITS4 primers, and fragments of both β-tubulin and translation elongation factor 1-alpha (EF1-α) genes using Bt2a-Bt2b and EF1728F-EF1986R primers, respectively, were sequenced and analyzed using BLASTn query. Accession numbers of gene sequences submitted to GenBank were KF479465 to 67 for ITS region; KF479468 to 70 for β-tubulin; and KF479471 to 73 for EF1-α. All sequences were 99 to 100% identical to reference isolate CMW7024 (4) of N. mangiferae (GenBank Accession Nos. AY615185, AY615172, and DQ093221). For each fungal isolate, pathogenicity tests were conducted on mango trees using six randomly selected, healthy mango inflorescences at full bloom using two trees per cultivar. Both needle-wounded and unwounded inflorescences were inoculated with 5-mm diameter mycelial disks from 8-day-old cultures grown on APDA. Inflorescences were incubated in clear plastic bags for 8 days under field conditions. Controls were treated with APDA disks only. Inflorescences on ‘Irwin’ turned brown with necrosis extending from the rachis to flowers. Mycelial growth and inflorescence blight was observed with lesions ranging from 2 to 5 cm in length. On ‘Haden,’ the rachis tissues turned brown and necrotic with lesions ranging from 1.5 to 2 cm long and without mycelial growth. N. mangiferae was re-isolated from all diseased inflorescences, and no symptoms developed on controls, which fulfilled Koch's postulates. The test was repeated once. N. mangiferae was associated with blossom blight in Australia and South Africa (2,3). This is the first report of N. mangiferae causing rachis necrosis and inflorescence blight on mango in Puerto Rico. N. mangiferae belongs to a complex of pathogens causing inflorescence blight and rachis necrosis and, therefore, effective management of this important disease complex must involve control of this pathogen. References: (1) P. W. Crous et al. Stud. Mycol. 55:235, 2006. (2) G. I. Johnson et al. Ann. Appl. Biol. 119:465, 1991. (3) J. H. Lonsdale and J. M. Kotzé. Acta Hortic. 341:345, 1993. (4) A. J. L. Phillips. Key to the various lineages in “Botryosphaeria” Version 01 2007. Last retrieved 5 February 2014 from http://www.crem.fct.unl.pt/botryosphaeria_site/key.htm .

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First Report of Valsa leucostoma Causing Valsa Canker of Pyrus communis (cv. Duchess de' Angouleme) in China

Plant Disease ◽

10.1094/pdis-07-13-0704-pdn ◽

2014 ◽

Vol 98 (3) ◽

pp. 422-422 ◽

Cited By ~ 1

Author(s):

M. X. Zhang ◽

L. F. Zhai ◽

W. X. Xu ◽

N. Hong ◽

G. P. Wang

Keyword(s):

Pyrus Communis ◽

Liaoning Province ◽

Post Inoculation ◽

Internal Transcribed Spacer Region ◽

Canker Disease ◽

First Report ◽

Agar Plug ◽

Valsa Canker ◽

Pear Trees ◽

Β Tubulin

Pear is a popular fruit in the world market, and has been widely cultivated in China. Since 2008, a severe canker disease has consistently been observed on 20-year-old pear trees (Pyrus communis cv. Duchess de' Angouleme) grown in a nursery in Xingcheng, Liaoning Province, China. Observed symptoms include brown elongated ulcerative lesions (more than 20 cm in length in general), with red brown conidia produced on wet lesions. Reductions in tree vigor and yield were observed for infected trees. Tree mortality was observed for severe infections. To diagnose the pathogen, 15 canker samples were collected from five pear trees in April, 2012. Bark pieces (3 to 5 mm) taken from the border of healthy and diseased tissue were surface-disinfected with 0.1% mercury bichloride and 75% ethanol for 45 s, and placed on potato dextrose agar (PDA) medium at 25°C in darkness. Fungal colonies with a common colony morphology were consistently recovered from three samples. These fungal colonies were initially white, becoming olive green in 3 days. Conidia produced on colonies were hyaline, allantoid, and single-celled with average length × width of 6.04 (5.43 to 6.59) × 0.65 (0.51 to 0.73) μm, which were consistent with descriptions of Valsa leucostoma (1). Genomic DNA was extracted from a representative isolate F-LN-32b, and subjected to PCR amplification of the internal transcribed spacer region (ITS), β-tubulin gene, and EF1 gene using the primer pairs ITS1/ITS4, Bt2a/Bt2b and EF1-728F/EF1-986R (3), respectively. Sequence alignment of the amplified fragments with the deposited data in NCBI showed that sequences of EF1, ITS, and β-tubulin (GenBank Accession No. KF293296 to KF293298, respectively) of isolate F-LN-32b had the highest similarity of 99% to those of V. leucostoma strain 32-2w (JQ900340, JN584644, and JQ900374), and suggested that isolate F-LN-32b is a V. leucostoma strain. Pathogenicity tests was carried out by placing a 5-mm-diameter, 2-day-old mycelium agar plug of isolate F-LN-32b onto a punched bark hole of a detached 1-year-old pear shoot after it was surface disinfested with ethanol. Inoculated shoots were incubated at 25°C in plastic containers covered with plastic film. Pathogenicity assays were conducted on 18 pear varieties (cvs. Qiuyue, Jinshui 2, Hohsui, Huali 1, Cuiguan, Shinseiki, Xuehua, Dangshansu, Zaosu, Hongxiangsu, Yuluxiang, Nanguoli, Xizilv, Bartlett, Huanghua, Huashan, Duchess de' Angouleme, and Packham's) collected from a nursery in Wuhan, Hubei Province, China. Six shoots were inoculated for each variety and the assay was conducted three times. All inoculated shoots developed the typical canker symptoms after 6 days post inoculation (dpi) and sporulated at 25 dpi while the control shoots inoculated with non-colonized PDA plugs remained asymptomatic. Isolates recovered from inoculated samples were of the same morphology and ITS sequence as F-LN-32b. Based on these results, V. leucostoma was determined as the pathogen responsible for the Valsa canker disease on pear. Valsa mali var. pyri was identified as the only pathogen causing Valsa canker disease on pear in China (2). To our knowledge, this is the first report of V. leucostoma causing a canker disease on pear in China. References: (1) G. C. Adams et al. Australas. Plant Pathol. 35:521, 2006. (2) X. L. Wang et al. Mycologia 103:317, 2011. (3) T. J. White et al. Pages 315-322 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

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First Report in Hawai'i of Xanthomonas citri pv. mangiferaeindicae Causing Bacterial Black Spot on Mangifera indica

Plant Disease ◽

10.1094/pdis-11-12-1071-pdn ◽

2013 ◽

Vol 97 (9) ◽

pp. 1244-1244 ◽

Cited By ~ 1

Author(s):

J. Yasuhara-Bell ◽

A. S. de Silva ◽

A. M. Alvarez ◽

R. Shimabuku ◽

M. Ko

Keyword(s):

Mangifera Indica ◽

Infected Plant ◽

Black Spot ◽

The United States ◽

Negative Control ◽

Sterile Water ◽

Xanthomonas Citri ◽

Mango Fruit ◽

First Report ◽

16S Rdna Pcr

Bacterial black spot of mango (Mangifera indica) caused by Xanthomonas citri pv. mangiferaeindicae (Xcm), is an economically important disease in tropical and subtropical areas (3). Xcm can infect a wide range of mango cultivars and induces raised, angular, black leaf lesions, sometimes with a chlorotic halo. Fruit symptoms appear as small, water-soaked spots on the lenticels that become star-shaped, erumpent, and exude an infectious gum (3). The bacterium can also cause latent infections (2). Immature mango fruit with black spots on the epidermis were collected in August 2012 from mango trees of the cvs. Raposa and Pirie at a residence in Pukalani, Hawai'i, on the island of Maui. Similar symptoms were seen on a tree of the mango cv. Common (also known as ‘Spanish’ or ‘Lahaina’) at a nearby golf course. Mango fruit with black lesions, and leaves showing black lesions with yellow halos, were collected in August 2012 from mango trees of the cv. Haden at a residence in Kaimuki, Hawai'i, on the island of O'ahu. Xanthomonas-like bacterial colonies were isolated on TZC agar. Suspect colonies were non-pigmented on YDC agar. A fruit strain of the bacterium from Maui (A6081A) and a strain from each of a fruit (A6081B) and a leaf (A6082) from O'ahu were each gram-negative, oxidative, positive for both starch and esculin hydrolysis, and negative for nitrate reduction, resulting in presumptive identification as a Xanthomonas sp. The three strains were further characterized by Microlog (Biolog, Inc. Hayward, CA), which showed the closest match with X. campestris. In addition, 16S rDNA PCR assays showed the closest match (99% similarity) with X. citri strains, and RIF marker analysis of dnaA (4) grouped the three strains with Xcm strain LMG 941 (Accession No. CAHO01000002.1). Hypersensitivity responses typical of xanthomonads were observed when these strains were infiltrated into tobacco leaves, whereas no response was observed using sterile water. Leaves of 3-week-old mango seedlings were infiltrated using 10 μl (~108 CFU/ml) of each strain suspended in sterilized water (six to eight inoculations per leaf, four leaves per plant, and three replicate plants per strain). The negative control treatments consisted of inoculation with sterile water, as well as an incompatible pathogen, X. hortorum pv. vitians (A6076), isolated from lettuce. Typical symptoms of bacterial black spot were observed for all strains assayed approximately 2 weeks after inoculation. No lesions were observed on the negative control plants. Koch's postulates were satisfied following reisolation and identification of the Xanthomonas strains from the infected plant tissues, using the biochemical and PCR methods described above. Results for strains from the two islands confirmed published descriptions of the pathogen, indicating that the pathogen causing symptoms on these mango trees is Xcm (1). Cultures and infected plant samples were sent to USDA APHIS and CPHST NPGLB facilities where this identification was confirmed. To our knowledge, this is the first report of bacterial black spot of mango in Hawai'i or anywhere in the United States. It is unknown whether this disease is a new occurrence or has not been reported previously. The origin of the primary inoculum is unknown. References: (1) B. Manicom and F. Wallis. Int. J. Syst. Bacteriol. 34:77, 1984. (2) O. Pruvost et al. Microbial Ecol. 58:928. (3) O. Pruvost et al. Plant Dis. 95:774, 2011. (4) K. Schneider et al. PLoS 6:e18496, 2011.

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