First Report of Leaf Anthracnose Caused by Colletotrichum karstii of Piper nigrum in Yunnan Province, China

Pepper (Piper nigrum L.) is one of the economically important spice crops of China and mainly grown in the Hainan and Yunnan provinces. In January 2021, the classic anthracnose lesions were observed on pepper leaves at a plantation (24°57’50"N, 98°53’00"E) in Baoshan city, Yunnan, China. Most of the diseased spots occurred at the tips and margins of the old pepper leaves. Lesions were grayish brown or pale white with a slight yellow halo, concentric whorl black dots or scattered black dots were observed on the leaves spots sometimes (Fig. 1). Five symptomatic leaves from different parts of the field were sampled for pathogen isolation. Lesion tissues removed from the border between symptomatic and healthy tissue were surface sterilized in 75% ethanol, then air-dried, plated on potato dextrose agar medium plates (PDA), and incubated in a 12-h photoperiod at 28℃. Similar fungal colonies developed from all plated tissues after 5 days. And five isolates from different leaves (one isolate per leaf) were sub-cultured using the single-spore method. The colonies appeared white, cottony, aerial mycelium dense and slow-growing (mean 1.01 mm day–1) on PDA plates in 6 days. Conidia were short-cylindric, straight, sometimes slightly constricted near the center, ends broadly rounded, measuring 11.05 to 14.43 × 3.78 to 6.08 µm (average = 12.03 × 5.48 µm, n=200). Appressoria were single, subglobose to elliptic, light brown to dark black. Among them, genomic DNA of two isolates (21HJ0301-1 and 21HJ0301-2) were extracted from mycelium and used as a template for molecular identification. The internal transcribed spacer (ITS) region of ribosomal DNA, and partial sequence of chitin synthase (CHS-1), actin (ACT) and glyceraldehydes-3-phosphate dehydrogenase (GAPDH) gene regions were amplified with primer pairs ITS1/ITS4, CHS-79F/CHS-354R, ACT-512F/ACT-783R, GDF/GDR, respectively (Weir et al. 2012). These four gene sequences were deposited in GenBank (Accession No. MZ725047 and MZ725048 for ITS, MZ733415 and MZ733416 for GAPDH, MZ733408 and MZ733409 for ACT, MZ733422 and MZ733423 for CHS-1). A multilocus phylogenetic analysis performed with the reference sequences revealed that both 21HJ0301-1 and 21HJ0301-2 isolates clustered with C. karstii (Fig.2). Based on morphology and molecular results, isolates were confirmed to be C. karstii. Pathogenicity tests were carried out on potted seedlings in the greenhouse, six healthy leaves per isolate were inoculated with six-day-old cultures of C. karstii mycelial discs of 5 mm in diameter after being wounded with a needle or non-wounded. Control leaves were inoculated with PDA agar. Inoculated plants were incubated under high relative humidity at room temperature. Anthracnose symptoms appeared within 5 days using non-wounded or wounded inoculation methods. All control leaves remained asymptomatic. The fungus was re-isolated from inoculated leaves fulfilling Koch’s postulates, but not on controls. C. karstii has a wide range of hosts, such as rubber tree, tea-oil tree, chili, and some other plants belonging to the family Orchidaceae in China (Cai et al. 2016; Jiang and Li 2018; Diao et al. 2017; Yang et al. 2011). To the best of our knowledge, this is the first report of C. karstii on Piper nigrum in China. This report will help us to recognize the anthracnose disease of Piper nigrum and establish a foundation for future studies on C.karstii to address effective management strategies.

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First Report of Ceratocystis changhui Causing Postharvest Fruit Rot of Peach in Kunming, China

Plant Disease ◽

10.1094/pdis-07-20-1492-pdn ◽

2020 ◽

Author(s):

Xue Li ◽

Ruiqi Zhang ◽

Kecheng Xu ◽

Jie Li ◽

Yu Zhang ◽

...

Keyword(s):

Phylogenetic Trees ◽

Prunus Persica ◽

Management Strategies ◽

Its Region ◽

Its Sequences ◽

Fruit Rot ◽

Single Spore ◽

Early Maturity ◽

Potato Dextrose Agar Medium ◽

First Report

The peach (Prunus persica (L) Batsch) is a predominant commercially grown stone fruit in China (Lee et al. 1990). Ceratocystis changhui is an aggressive pathogen causing typical black rot symptoms on corms of taro (Colocasia esculenta) (Liu et al. 2018), it has not been reported on other hosts. During the summer and autumn of 2013, a postharvest fruit rot disease was observed on several peaches at a farmer's market (N 25°02′; E 102°42′) in Kunming City, Yunnan Province, China. The incidence of the disease varied from 5 to 20%. Necrotic spots were first observed on the infected peach fruit (Prunus persica cv. shuimitao). The spots enlarged gradually and developed into a brown, water-soaked and rotted lesion. Eventually, the whole fruit became soft, rotted and covered with a gray-brown mycelium (Fig. 1 A, B). The isolates were obtained from the symptomatic tissues incubated on slices of fresh carrot root (Moller et al. 1968). After 5 to 10 days of incubation, perithecia and mycelium were observed growing on carrot slices. Spore masses were removed from the apices of perithecia, transferred to potato dextrose agar medium (PDA) and incubated at 25°C for 5 to 10 days, followed by single-spore isolation. All eight single-spore isolates from peach fruits obtained in this study were deposited in the State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan Agricultural University, China. In culture, mycelium was initially white, gradually turned to greyish-green or brown (Fig. 1E, F). Measurements were made 7 days after the formation of perithecia. Perithecia (Fig. 1G) were black, globose, 185.71 to 305.56 μm × 142.86 to 264.29 µm and showed a long black neck, 600 to 957.14 µm (Fig. 1H). Ascospores (Fig. 1I) were helmet-hat shaped and 2.86 to 6.67 µm ×3.81 to 4.76 µm. Cylindrical conidia (Fig. 1J) 6.67 to 38. 95 µm × 2.86 to7.62 µm were observed. Chlamydospore (Fig. 1K), 8.57 to 13.33 μm × 5.71 to 9.52 μm, were ovoid or obpyriform, smooth. To further verify pathogen identity the internal transcribed spacer (ITS) region of rDNA was amplified using primers ITS1F and ITS4 (Thorpe et al. 2005), and the total genomic DNA from the mycelia of ﬁve isolates was extracted using a CTAB method (Lee &Taylor 1990). The nucleotide sequences have been blasted and deposited in the GenBank database. Analysis of the ITS sequences from the isolates T1-1yp, T1-2yp, T2-1yp (GenBank accession no. KY580895-KY580897) showed 99% to 100% similarity with isolates C. changhui CMW43272 (KY643886), CMW43281 (KY643884), CMW46112 (KY643891) and CMW46113 (KY643892) from taro in China. Phylogenetic trees based on the maximum-likelihood (ML) method were constructed using MEGA 7. ITS sequences of other Ceratocystis spp. were attained from NCBI for comparative analysis (Liu et al. 2018), and Davidsoniella virescens (CMW11164) served as outgroup. The robustness of ML tree was evaluated with 1,000 bootstrap (BS) values. The pathogen was identified as C. changhui based on the phylogenetic analysis (Fig. 2). Three isolates (T1-1yp, T1-2yp, T2-1yp) were used for pathogenicity. Nine Prunus persica cv. yingzuitao fruits at early maturity (8 points out of 10) were wound inoculated with 200μL conidia suspension of the fungus (approximately 2.0 × 106 conidia / mL). Degreasing cotton dipped in sterile water was used to raise the humidity in preservation boxes. Boxes were incubated for 10 days at 25°C. Three peaches as controls were treated only with sterile distilled water in the same way. Symptoms of sunken lesions and fruit rot were observed two days after inoculation, and measured at 1.8 to 3.2 cm from the inoculation point within 5 days (Fig. 1C: right, D). The same pathogen was re-isolated from them confirming Koch’s postulates. Control peaches remained symptomless. This fungus was morphologically and phylogenetically identified as C. changhui. To our knowledge, this is the first report of C. changhui on postharvest peach in Yunnan, China. The disease will affect quality and taste of peach, so it is critical to deploy appropriate management strategies to limit the fungus spread.

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First Report of Neofusicoccum australe and N. luteum Associated with Canker and Dieback of Quercus robur in Portugal

Plant Disease ◽

10.1094/pdis-10-12-0955-pdn ◽

2013 ◽

Vol 97 (4) ◽

pp. 560-560 ◽

Cited By ~ 11

Author(s):

C. Barradas ◽

A. Correia ◽

A. Alves

Keyword(s):

Quercus Robur ◽

Elongation Factor ◽

Aerial Mycelium ◽

Its Region ◽

Deciduous Tree ◽

Pedunculate Oak ◽

Single Spore ◽

Canker Disease ◽

First Report ◽

Wide Range

Quercus robur L., commonly known as “pedunculate oak,” is a long-lived deciduous tree native to most of Europe. It is of great ecological and forestry importance. It is also commonly cultivated as an ornamental tree in parks and gardens. Since 2009 and most likely related to increased drought periods, diseased ornamental trees have been observed in the campus of the University of Aveiro, Portugal. More than 50% of the trees are already damaged by the disease. The symptoms included twig and branch dieback and sunken necrotic bark lesions that could progress to the trunk, resulting in the death of large sections of the tree. Ascomata and conidiomata typical of Botryosphaeriaceae were observed on branches of symptomatic trees. Ascospores were hyaline, aseptate, ovoid to fusoid, and conidia were hyaline, aseptate, smooth, thin-walled, and fusiform with base truncate. Single spore isolates were obtained from samples. In culture, single ascospore isolates produced conidia similar to the ones found on the host. Diseased branch tissues were surface sterilized with 5% NaOCl, plated on potato dextrose agar (PDA), and incubated at 25°C. Fungal isolates recovered produced white aerial mycelium that darkened with age becoming grey to dark grey and conidia that were similar in all aspects to the ones produced by single spore isolates. All isolates produced on PDA a yellow pigment that diffused into the agar and disappeared after 6 to 7 days. Morphological and cultural aspects of the isolates were similar to the species Neofusicoccum luteum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips and N. australe (Slippers, Crous & M.J. Wingf.) Crous, Slippers & A.J.L. Phillips (2). Enterobacterial Repetitive Intergenic Consensus (ERIC) PCR fingerprinting divided the isolates into two groups (1). Two isolates (CAA352 and CAA392), one from each group, were selected for further identification by sequencing of the internal transcribed spacer (ITS) region of the rDNA and part of the elongation factor 1-alpha gene (EF1-α) (Accession No. ITS: JX975212 JX975213; EF1-α: JX975210 JX975211). BLAST analysis showed that ITS and EF1-α sequences from group 1 and 2 had 99 to 100% similarity to reference cultures (including ex-type) of N. luteum and N. australe, respectively. To confirm pathogenicity and fulfill Koch's postulates, six 2-year-old seedlings of Q. robur were artificially infected with isolates CAA352 and CAA392 and kept at approximately 20 to 25°C. A shallow wound was done with a scalpel on the basal part of the stem of each seedling, a bark portion was removed aseptically and a PDA disc (0.5 cm) of an actively growing culture was placed on the wound. Control seedlings received sterile PDA discs. The inoculation site was wrapped in Parafilm to prevent desiccation. Within 8 weeks, infected seedlings developed canker lesions associated with vascular necrosis around the inoculation point. A third of the seedlings died and developed abundant pycnidia on the stem. Control seedlings remained symptomless. Both pathogens were successfully reisolated from the infected tissue. N. luteum and N. australe are increasingly reported as causing diseases to a wide range of woody hosts of economic and forestry importance (3). To our knowledge, this is the first report of both species causing dieback and canker disease on Q. robur. References: (1) A. Alves et al. Res. Microbiol. 158:112, 2007. (2) P. W. Crous et al. Stud. Mycol. 55:235, 2006. (3) B. Slippers et al. Fungal Biol. Rev. 21:90, 2007.

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First Report of Colletotrichum siamense Causing Daylily (Hemerocallis citrina) Anthracnose in China

Plant Disease ◽

10.1094/pdis-04-21-0817-pdn ◽

2021 ◽

Author(s):

Taixiang Chen ◽

Han Lin Yue ◽

Yong Xin Nie ◽

Wanrong Wei

Keyword(s):

Disease Incidence ◽

Management Strategies ◽

Its Region ◽

Culture Collection ◽

Gansu Province ◽

Perennial Herb ◽

Potato Dextrose Agar Medium ◽

First Report ◽

Colletotrichum Siamense ◽

Hemerocallis Citrina

Daylily (Hemerocallis citrina Baroni) is a perennial herb whose flowers are commonly used in traditional Chinese cuisine. It is commercially cultivated in the Loess plateau of Gansu province, China. From July to October 2020, necrotic lesions were observed on the foliage of daylily plants in Huan County, Gansu, China, with an average disease incidence of 90%, and 52 to 86 disease index across four fields (approximate 6 hectares). Lesions were fusiform or nearly fusiform yellowish-brown spots of different sizes and a yellow irregular border. Older lesions were almost dark brown that often coalesced and expanded to cover the entire leaves. Thirty-four samples were collected from plants with typical foliar symptoms. Symptomatic tissues were excised from the margins of the lesions and sterilized with 75% ethanol for 20 s and 0.1% NaClO for 2 min, rinsed with sterilized water four times, dried on sterile paper towels, and cultured on Potato Dextrose Agar medium at 25°C for 7 days. A total of 34 fungal isolates with 100% isolation frequency were obtained and characterized. Colonies were white, becoming pale brown with age, reverse turned grayish black with age and irregular pale yellowish borders on the reverse side. Conidia (n=50) were hyaline, one-celled, subcylindrical with obtuse to slightly rounded ends, of 12-18.5×3.5-6 µm in size, (avg. 15.5×4.8 µm). The isolates were designated as K2010301 (51-54) and deposited in the Microbiological Culture Collection Center at College of Pastoral Agriculture Science and Technology, Lanzhou University (China). For fungal identification to species level, genomic DNA of a representative isolate (isolate MG) was extracted. Internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase-1 (CHS-1) and beta-tubulin (TUB2) were amplified using V9G/ITS4, GDF1/GDR1, CHS-354R/CHS-79F, and T1/Bt-2b primer sets (Damm et al., 2012), respectively, and deposited in GenBank under accession numbers MW811458, MW836582, MW836581, and MW836584. BLASTn showed higher than 99% identity with Colletotrichum siamense (GenBank: KP703350 (ITS), MN884050 (GAPDH), MN894598 (CHS-1), and KX578815 (TUB2)). A Bayesian inference analysis of the four concatenated loci showed that isolate MG grouped in the C. siamense clade. Pathogenicity tests were performed by spraying a spore suspension (1×105 conidia/mL) of a 10-day-old culture of isolate “MG” onto 3 healthy and asymptomatic daylily plants. Three control plants were only sprayed with the same volume of sterile distilled water. The inoculated plants were covered with black plastic bags for 2 days to maintain high relative humidity. Anthracnose symptoms resembling those observed in the field developed after 7 days on all inoculated plants, while no symptoms were observed on the control plants. The fungus was reisolated and identified as C. siamense based on morphological features and DNA sequence analysis, fulfilling Koch’s postulates. It has been demonstrated that C. liliacearum (Zhuang, 2005), C. gloeosporioides, and C. spaethianum (Yang et al., 2012) are anthracnose pathogens of H. citrina. To our knowledge, this is the first report of C. siamense causing daylily anthracnose worldwide. This fungal pathogen represents a severe threat and has the potential to cause yield losses of daylily, so further studies should focus on epidemiology and effective management strategies of this disease.

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First Report of Strawberry Dieback Caused by Lasiodiplodia theobromae

Plant Disease ◽

10.1094/pdis-11-13-1192-pdn ◽

2014 ◽

Vol 98 (11) ◽

pp. 1579-1579 ◽

Cited By ~ 6

Author(s):

A. Yildiz ◽

K. Benlioglu ◽

H. S. Benlioglu

Keyword(s):

Management Practice ◽

Fungal Growth ◽

Aerial Mycelium ◽

Plant Diseases ◽

Botryodiplodia Theobromae ◽

Single Spore ◽

Lasiodiplodia Theobromae ◽

First Report ◽

Wide Range ◽

Crown Tissue

With a typical Mediterranean climate, Aydin is the third largest strawberry-producing province, responsible for 13% of the overall strawberry production in Turkey. Strawberries (Fragaria × ananassa Duchesne) are mainly grown in raised, plastic-mulched beds under tunnels and soil solarization is the most effectively used management practice to control soil-borne pathogens. During October 2011 and 2012, 2 months after planting, wilting and collapse of plants were observed on commercial strawberry (cv. strawberry Festival) fields in Sultanhisar town of Aydin Province. Eleven percent of the plants were wilted and died. Symptomatic plants exhibited blackened necrotic discoloration of roots and in the cross section of crowns. A fungus was consistently isolated from pieces of infected tissue cut aseptically from the crowns and placed on potato dextrose agar. Fungus produced white colonies and later turned olivaecious black with dense aerial mycelium after 4 to 5 days incubation at 27°C. Dark brown to black pycnidia that formed on 20- to 30-day-old pure cultures under daylight conditions produced abundant conidia that were two-celled, thick-walled, and oval shaped with longitudinal striations. Single spore isolates from 12 samples were obtained and stored for further identification. The average size of 300 conidia was 25.42 ± 2.12 × 12.87 ± 1.08 μm. The morphology of the fungus was similar to Lasiodiplodia theobromae (Pat.) Griff. & Maubl. (syn. Botryodiplodia theobromae Pat.). To confirm the identity of the isolates, the internal transcribed spacer (ITS) region of ribosomal DNA and the elongation factor 1-alpha gene were amplified with the universal ITS1/ITS4 and EF1-688F/EF1-1251R (1) primers, respectively. The amplicons from 12 isolates were commercially sequenced at Macrogen (Korea) and were deposited in GenBank under consecutive accession numbers KF910369 to KF910380 and KJ641536 to KJ641547. Sequence comparison and phylogenetic analysis revealed that all 12 isolates were closely related and belonged to L. theobromae. Pathogenicity tests were performed by the toothpick technique (2) under greenhouse conditions (28°C, 14/10-h day/night, 70% RH) on potted strawberry plants (cv. strawberry Festival). Toothpicks carrying fungal growth taken from 1-week-old corn meal agar cultures of the tested isolates was placed into the basal crown tissue of the plants by piercing about 5 mm depth. Six plants were inoculated for each isolate and six were treated with sterile toothpick for control. All inoculated plants developed wilting and dieback symptoms resembling those of naturally infected plants within 2 to 3 weeks of incubation. All plants inoculated with the tested isolates collapsed after 4 weeks and showed discoloration of internal crown tissue. Control plants did not exhibit any disease symptoms, and crown tissue was symptomless. L. theobromae was successfully re-isolated from lesions of all inoculated plants. L. theobromae has been reported to cause cankers and dieback in a wide range of hosts in tropical and subtropical regions of the world (3). To the best of our knowledge, this is the first report of L. theobromae causing dieback on strawberry plants. References: (1) A. Alves et al. Fungal Divers. 28:1, 2008. (2) M. E. A. El-Morsi and I. A. Ibrahim. Wudpecker J. Agric. Res. 1:215, 2012. (3) E. Punithalingam. Plant diseases attributed to Botryodiplodia theobromae Pat. J. Cramer, Vaduz, 1980.

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First Report of Cankers and Dieback Caused by Neofusicoccum mediterraneum and Diplodia corticola on Grapevine in Spain

Plant Disease ◽

10.1094/pdis-05-11-0429 ◽

2011 ◽

Vol 95 (10) ◽

pp. 1315-1315 ◽

Cited By ~ 7

Author(s):

C. Pintos Varela ◽

V. Redondo Fernández ◽

O. Aguín Casal ◽

J. P. Mansilla Vázquez

Keyword(s):

Average Length ◽

Aerial Mycelium ◽

Its Region ◽

Single Spore ◽

Mycelium Growth ◽

First Report ◽

Tubulin Genes ◽

Agar Plug

In November 2010, four grapevine plants of cv. Crimson from a vineyard located in Sevilla (south Spain) revealed trunk cankers. Several pathogens were isolated, including Cylindrocarpon liriodendri (2), Phaeoacremonium aleophilum (2), Pleurostomophora richardsiae, Neofusicoccum parvum, and Botryosphaeria dothidea (2). Among Botryosphaeriaceae fungi isolated on potato dextrose agar (PDA) were two types that did not fit the above mentioned species. Isolates of type 1 produced an abundant, gray mycelium with a diurnal zonation that gradually became dark olivaceous. Mycelium growth occurred from 5 to 37°C with an optimum at 28°C. Conidia were hyaline, fusiform, aseptate, thin walled, but gradually became obscured and septate with age, and measured (18.4-) 21.4 (-24.3) × (4.2-) 5.5 (-7.2) μm with a length/width (L/W) ratio of 4.0 ± 0.5 (n = 100). Isolates of type 1 were identified as N. mediterraneum (3). Single-spore cultures of type 2 developed a whitish, dense, aerial mycelium and remained white up to 10 days on PDA and darkened to gray thereafter. Mycelium growth occurred from 3 to 37°C with an optimum at 29 to 30°C. Conidia were hyaline, aseptate, thick walled, oblong to cylindrical, sometimes becoming light brown and one or two septate after discharge, and measured (24.6-) 30.2 (-42.8) × (10.9-) 14.3 (-18.6) μm with a L/W ratio of 2.1 ± 0.2 (n =100). Isolates of type 2 were identified as Diplodia corticola (1). Nucleotide sequences of the ribosomal internal transcribed spacer (ITS) region and the -tubulin genes were used to confirm the identifications through BLAST searches in GenBank. Comparison of the sequences of types 1 and 2 showed 99 to 100% homology with N. mediterraneum (HM443604 (4) and GU251836) and D. corticola (AY268421 (1) and EU673117), respectively. Representative sequences of N. mediterraneum (JF949757 and JF949756) and D. corticola (JF949758 and JF949759) were deposited in GenBank. The pathogenicity of one representative isolate of each of N. mediterraneum and D. corticola was confirmed by inoculating 10 detached grapevine canes (averaging 12 mm in diameter and 30 cm long) per isolate. A shallow wound was made with a scalpel on the internodes. A colonized 6-mm agar plug, from the margin of an actively growing colony, was inserted in every wound and sealed with Parafilm. Ten grapevine canes controls received only sterile PDA agar plugs. Canes were maintained at 25°C and 70% humidity. After 5 weeks, all inoculated canes developed cankers and pycnidia around the inoculation site. Vascular necroses that developed on the inoculated canes were an average of 28.6 mm for N. mediterraneum and 27.7 mm for D. corticola. One-way analysis of variance and Tukey's test confirmed significant differences in the extent of vascular necroses. The average necroses length in the inoculated canes was significantly greater (P < 0.05) than the average length of discoloration induced by the simulated inoculation process in the control. Both pathogens were reisolated from all inoculated plants but not from controls. To our knowledge, this is the first report of N. mediterraneum and D. corticola as pathogens on grapevine in Spain. References: (1) A. Alves et al. Mycologia 96:603, 2004. (2) A. Aroca and D. Gramaje et al. Eur. J. Plant. Pathol. 126:165, 2010. (3) P. W. Crous et al. Fungal Planet. No. 19, 2007. (4) F. P. Trouillas et al. Plant. Dis. 94:1267, 2010.

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First Report of Diaphorte terebinthifolii causing leaf spot on Pleoroma fotherghillae in Brazil

Plant Disease ◽

10.1094/pdis-11-20-2508-pdn ◽

2021 ◽

Author(s):

Alexandre Claus ◽

Wagner V. Pereira ◽

Louise Larissa May De Mio

Keyword(s):

Leaf Spot ◽

Economic Value ◽

Aerial Mycelium ◽

Its Region ◽

Botanical Garden ◽

Control Treatment ◽

Transparent Plastic ◽

Potato Dextrose Agar Medium ◽

First Report ◽

Leaf Spots

Pleoroma fotherghillae, also known as “princess flower”, is an ornamental species native to Brazil and naturalized in several countries (Faravani et al. 2007). P. fotherghillae has a high economic value, with an ornamental and landscape application (Nienow et al. 2010). In September 2018, leaf spots were observed in approximately 80% of the 50 P. fotherghillae plants grown in a nursery in the municipality of Curitiba-Paraná, Brazil. The spots were round-shaped, with a necrotic brown center and a reddish-brown halo, ranging from 1 to 4 mm in diameter. High leaf fall was observed among plants presenting a higher severity. Symptomatic leaves fragments were collected and disinfected as described by (Pereira et al. 2019). The fragments were transferred to a potato dextrose agar medium supplemented with streptomycin sulfate and incubated at 24 ± 1ºC with a photoperiod of 12 h for 7 days. Four monosporic cultures were obtained from colonies isolated. The isolates had a grayish-white cottony aerial mycelium and reverse olive-yellow with black dots. The colonies reached approximately 60 mm in diameter, forming globular and conical pycnidia, brown to black in color with white or cream globular conidial mass. Beta conidia were hyaline, smooth, curved to the size of 19 - 25 x 1 – 1.5 μm (n = 50). No alpha nor gamma conidia were observed. The characteristics are similar to the description of Diaporthe terebinthifolli (Gomes et al. 2013). The total genomic DNA of a representative isolate, LEMIDPRPf-19-02, was extracted for amplification and sequencing of the internal transcribed spacer (ITS) region and partial of the Tubby (TUB) and thyrotroph embryonic factor (TEF) genes. The sequences of the ITS (No MN415990.1), TUB (No MW505549), and TEF (No MW505550) genes were deposited in GenBank. BLAST analysis showed similarity above 99% with D. terebinthifolli sequences (KC343219.1, KC344187.1, and KC343945.1). The multigene phylogenetic analysis, based on Bayesian Inference, grouped the isolate in a clade with other sequences of Diaporthe terebinthifolii. Four healthy plants of P. fotherghillae about 5 months old, were used for pathogenicity testing. A suspension containing 105 conidia/ml was sprayed on the surface of the leaves of four plants to the point of runoff. The plants were covered with a transparent plastic bag for 24 hours. The leaves of four other plants received sterile distilled water and served as the control treatment. The plants were kept in a greenhouse at 20±5ºC. Necrotic lesions appeared 10 to 15 days after inoculation. No symptoms were observed in the control plants. The pathogen was reisolated from symptomatic leaves and had the same characteristics as the isolate LEMIDPRPf-19-02. A representative sample (MBM 331603) was deposited at the Museu do Jardim Botânico (Botanical Garden Museum) - Curitiba, Brazil. Diaporthe terebinthifolii was previously reported as endophytic in Brazil and Uruguay, isolated from Schinus terebinthifolius and Pyrus communis, respectively (Gomes et al. 2013; Sessa et al. 2017). To our knowledge, this is the first report of D. terebinthifolii causing leaf spot on P. fotherghillae in Brazil and worldwide.

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First report of Neopestalotiopsis protearum causing seed rot on Camellia oleifera in China

Plant Disease ◽

10.1094/pdis-12-20-2717-pdn ◽

2021 ◽

Author(s):

Jie Tang ◽

YiLin Du ◽

LiXiang Lai ◽

Qin Yang

Keyword(s):

Unsaturated Fatty Acids ◽

Aerial Mycelium ◽

Brown Rot ◽

Its Region ◽

Hunan Province ◽

Bootstrap Support ◽

Camellia Oleifera ◽

Single Spore ◽

First Report ◽

Seed Rot

Camellia oleifera, an evergreen small tree or shrub with high medicinal and ecological values, is mainly distributed in subtropical regions of China. Camellia oil obtained from Camellia oleifera seeds is rich in unsaturated fatty acids and unique flavors, and has become a rising high-quality edible vegetable oil in south of China (Zhuang 2008). The tea-oil tree Camellia oleifera plays important economic and ecological roles in Hunan province. During collecting trips, seeds of C. oleifera with disease symptoms have been observed in almost all oil-tea forests. In lab, the seeds can be infected by wounds and directly, however, wound infection is more rapid. In oil-tea forests, the wound of seed is often caused by external factors such as mechanical and insects. Symptomatic seeds exhibited brown rot symptoms with irregular, black spots, brown necrosis of the kernels, and accounted for 65% of the surveyed seeds (Fig. 1). Rotted seeds were surface-sterilized for 1 min in 75% ethanol, 3 min in 1% sodium hypochlorite, then rinsed for 2 min in sterile water and blotted on dry sterile filter paper. Discolored seed tissues were cut into pieces of 3 mm × 3 mm using a sterile scalpel, placed on potato dextrose agar (PDA) medium, and then incubated for 7 days at 25°C with a 12-h photoperiod. After 7 days of incubation, circular fungal colonies with dense aerial mycelium, produced black, wet spore masses. Four-septate conidia were ellipsoidal to obovoid, measuring 24 (22 to 26) × 6.5 (6 to 7) µm (n = 30). Conidia had three median cells, which were dark brown, with a single basal hyaline appendage, 4 (3.5 to 4.5) µm long, and two to four (usually three) apical hyaline appendages, 32 (27 to 35) µm long, similar to these recorded by Crous et al. (2011). Two single-spore isolates cultured on PDA medium were selected for DNA extraction. The ITS region was amplified using primers ITS5 and ITS4 (White et al. 1990). The partial translation elongation factor 1-alpha (tef1-α) gene region was amplified using primers EF1-728F (O'Donnell et al. 1998) and EF-2 (Carbone & Kohn 1999). The partial β-tubulin (tub2) was amplified using primers T1 and Bt2b (Glass & Donaldson 1995). The sequences of ITS (MW391815), tef1-α (MW398222), and tub2 (MW398223) were submitted to GenBank. BLAST analysis demonstrated that these sequences were 99%~100% similar to the sequences of ITS (MH553959), tef1-α (MH554377), and tub2 (MH554618) published for Neopestalotiopsis protearum. Phylogenetic analysis revealed that all the representative isolates recovered from symptomatic Camellia oleifera seeds showed 91% bootstrap support with Neopestalotiopsis protearum isolate in references (Fig. 2). Pathogenicity tests were conducted on 20 healthy seeds. We wounded the seeds by a sterilized needle on the middle position, and put the 5-mm-diameter agar plugs with actively grown mycelia (strain HNWC04) or pure PDA on the wound. We then covered the wounds with clean masking tape to prevent contamination and desiccation. After inoculation, the seeds were kept at 90 to 100% relative humidity at 25°C in a greenhouse for 3 weeks and monitored daily for lesion development. Twenty days after inoculation, all the seeds inoculated presented similar typical symptoms observed under natural conditions, whereas the control seeds showed no symptoms. Koch’s postulates were fulfilled by reisolating the same fungus and verifying its colony and morphological characters as Neopestalotiopsis protearum. To our knowledge, this is the first report of Neopestalotiopsis protearum causing oil-tea seed rot in China.

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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 Stem Canker Disease of Pitaya (Hylocereus undatus and H. polyrhizus) Caused by Neoscytalidium dimidiatum in Taiwan

Plant Disease ◽

10.1094/pdis-08-11-0689-pdn ◽

2012 ◽

Vol 96 (6) ◽

pp. 906-906 ◽

Cited By ~ 18

Author(s):

M. F. Chuang ◽

H. F. Ni ◽

H. R. Yang ◽

S. L. Shu ◽

S. Y. Lai ◽

...

Keyword(s):

New York ◽

Aerial Mycelium ◽

Its Region ◽

Stem Canker ◽

Canker Disease ◽

First Report ◽

Neoscytalidium Dimidiatum ◽

Succulent Plant ◽

Sterile Medium ◽

Hylocereus Undatus

Pitaya (Hylocereus undatus and H. polyrhizus Britt. & Rose), a perennial succulent plant grown in the tropics, is becoming an emerging and important fruit plant in Taiwan. In September of 2009 and 2010, a number of pitaya plants were found to have a distinctive canker on stems. The disease expanded quickly to most commercial planting areas in Taiwan (e.g., Pintung, Chiayi, and Chunghua). Symptoms on the stem were small, circular, sunken, orange spots that developed into cankers. Pycnidia were erumpent from the surface of the cankers and the stems subsequently rotted. After surface disinfestation with 0.1% sodium hypochloride, tissues adjacent to cankers were placed on acidified potato dextrose agar (PDA) and incubated at room temperature for 1 week, after which colonies with dark gray-to-black aerial mycelium grew. Hyphae were branched, septate, and brown and disarticulated into 0- to 1-septate arthrospores. Sporulation was induced by culturing on sterile horsetail tree (Casuarina equisetifolia) leaves. Conidia (12.79 ± 0.72 × 5.14 ± 0.30 μm) from pycnidia were one-celled, hyaline, and ovate. The internal transcribed spacer (ITS) region of ribosomal DNA was PCR amplified with primers ITS1 and ITS4 (2) and sequenced. The sequence (GenBank Accession No. HQ439174) showed 99% identity to Neoscytalidium dimidiatum (Penz.) Crous & Slippers (GenBank Accession No. GQ330903). On the basis of morphology and nucleotide-sequence identity, the isolates were identified as N. dimidiatum (1). Pathogenicity tests were conducted in two replicates by inoculating six surface-sterilized detached stems of pitaya with either mycelium or conidia. Mycelial plugs from 2-day-old cultures (incubated at 25°C under near UV) were inoculated to the detached stems after wounding with a sterile needle. Conidial suspensions (103 conidia/ml in 200 μl) were inoculated to nonwounded stems. Noninoculated controls were treated with sterile medium or water. Stems were then incubated in a plastic box at 100% relative humidity and darkness at 30°C for 2 days. The symptoms described above were observed on inoculated stems at 6 to 14 days postinoculation, whereas control stems did not develop any symptoms. N. dimidiatum was reisolated from symptomatic tissues. To our knowledge, this is the first report of N. dimidiatum causing stem canker of pitaya. References: (1) P. W. Crous et al. Stud. Mycol. 55:235, 2006. (2) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, New York, 1990.

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First report of Diaporthe ueckerae causing stem canker on soybean (Glycine max L.) in Colombia

Plant Disease ◽

10.1094/pdis-04-21-0718-pdn ◽

2021 ◽

Author(s):

Nathali López-Cardona ◽

YUDY ALEJANDRA GUEVARA ◽

Lederson Gañán-Betancur ◽

Carol Viviana Amaya Gomez

Keyword(s):

Management Strategies ◽

Elongation Factor ◽

Morphological Characteristics ◽

Aerial Mycelium ◽

Stem Canker ◽

Growth Stages ◽

Internal Transcribed Spacer Region ◽

First Report ◽

Glycine Max L ◽

Soybean Plants

In October 2018, soybean plants displaying elongated black to reddish-brown lesions on stems were observed in a field planted to the cv. BRS Serena in the locality of Puerto López (Meta, Colombia), with 20% incidence of diseased plants. Symptomatic stems were collected from five plants, and small pieces (∼5 mm2) were surface sterilized, plated on potato dextrose agar (PDA) and incubated for 2 weeks at 25°C in darkness. Three fungal isolates with similar morphology were obtained, i.e., by subculturing single hyphal tips, and their colonies on PDA were grayish-white, fluffy, with aerial mycelium, dark colored substrate mycelium, and produced circular black stroma. Pycnidia were globose, black, occurred as clusters, embedded in tissue, erumpent at maturity, with an elongated neck, and often had yellowish conidial cirrus extruding from the ostiole. Alpha conidia were observed for all isolates after 30 days growth on sterile soybean stem pieces (5 cm) on water agar, under 25ºC and 12 h light/12h darkness photoperiod. Alpha conidia (n = 50) measured 6.0 – 7.0 µm (6.4 ± 0.4 µm) × 2.0 – 3.0 µm (2.5± 0.4 µm), were aseptate, hyaline, smooth, ellipsoidal, often biguttulate, with subtruncate base. Beta conidia were not observed. Observed morphological characteristics of these isolates were similar to those reported in Diaporthe spp. by Udayanga et al. (2015). DNA from each fungal isolate was used to sequence the internal transcribed spacer region (ITS), and the translation elongation factor 1-α (TEF1) gene, using the primer pairs ITS5/ITS4 (White et al. 1990) and EF1-728F/EF1- 986R (Carbone & Kohn, 1999), respectively. Results from an NCBI-BLASTn, revealed that the ITS sequences of the three isolates (GenBank accessions MW566593 to MW566595) had 98% (581/584 bp) identity with D. miriciae strain BRIP 54736j (NR_147535.1), whereas the TEF1 sequences (GenBank accessions MW597410 to MW597412) had 97 to 100% (330-339/339 bp) identity with D. ueckerae strain FAU656 (KJ590747). The species Diaporthe miriciae R.G. Shivas, S.M. Thomps. & Y.P. Tan, and Diaporthe ueckerae Udayanga & Castl. are synonymous, with the latter taking the nomenclature priority (Gao et al. 2016). According to a multilocus phylogenetic analysis, by maximum likelihood, the three isolates clustered together in a clade with reference type strains of D. ueckerae (Udayanga et al. 2015). Soybean plants cv. BRS Serena (growth stages V3 to V4) were used to verify the pathogenicity of each isolate using a toothpick inoculation method (Mena et al. 2020). A single toothpick colonized by D. ueckerae was inserted directly into the stem of each plant (10 plants per isolate) approximately 1 cm below the first trifoliate node. Noncolonized sterile toothpicks, inserted in 10 soybean plants served as the non-inoculated control. Plants were arbitrarily distributed inside a glasshouse, and incubated at high relative humidity (>90% HR). After 15 days, inoculated plants showed elongated reddish-brown necrosis at the inoculated sites, that were similar to symptoms observed in the field. Non-inoculated control plants were asymptomatic. Fungal cultures recovered from symptomatic stems were morphologically identical to the original isolates. This is the first report of soybean stem canker caused by D. ueckerae in Colombia. Due to the economic importance of this disease elsewhere (Backman et al. 1985; Mena et al. 2020), further research on disease management strategies to mitigate potential crop losses is warranted.

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