First Report of Mulberry Root Rot Caused by Lasiodiplodia theobromae in China

Mulberry (Morus alba L.) is an important cash crop and medicinal plant that has been cultivated for more than 5,000 years in China. The area of mulberry production in Guangxi Province is 45% of total production in China, with 1.3 million ha planted. In recent years, a mulberry root rot occurred in Heng County covering all the mulberry planting farms. Observations of 200 diseased plants were made. The xylem of infected roots first turned brown, and then became black followed by cortex rot. The xylem and cortex of infected roots were easily separated. The xylem of the stem of symptomatic plants was also brown and the bark was slightly darker than normal. Leaves of diseased plants turned yellow and wilted, but the wilted leaves remained on the affected branches for about 3 weeks. All affected branches and stem dried after a month. The affected area was 12,000 ha with incidences varying from 13 to 52%. About 8% of young mulberry trees died in severely infested orchards. The disease caused more than $3 million in losses within a year in Heng County alone. The causal fungus was isolated from xylem tissues of symptomatic roots of 62 mulberry plants with an isolation rate of 90%. Pathogenicity test was made by inoculating 5-month-old healthy mulberry plants with PDA plugs (5 × 5 mm) grown 5 days with viable mycelia of the fungus. Nine healthy plants were wounded on the roots with a sterile knife, and mycelial plugs of three Lasiodiplodia theobromae (Pat.) Griffon & Maubl isolates were placed on the wounds, covered with sterile moist cotton, and wrapped with Parafilm. Nine control plants were treated with PDA plugs. The test was repeated three times. All treated plants were kept in a greenhouse at ~28°C and 40% RH. After 3 days, the root xylem of inoculated plants turned brown and gradually became dark, similar to symptoms observed in the field. After 8 days, inoculated seedlings gradually wilted, and all the treated plants died after 11 days with leaves undetached. The fungus was re-isolated from all nine diseased plants and no symptoms were observed on the roots of control plants. The causal agent, of which conidia were dark brown, one-septate, thick walled, and ellipsoid with 4 or 6 vertical lines of dashes, 12.50 to 13.75 × 13.75 to 25.63 μm (n = 100), was identified as L. theobromae based on morphological characters described by Punithalingam (3) and sequences of the ITS region of rDNA using primers ITS1 and ITS4 and EF1-α using primers EF728F and EF986R. The ITS sequence (HG917932) was similar to the ITS sequences of AY640255 (CBS164.96) and AY236952 (CMW9074) in GenBank with identities of 98.8 and 99.8%, respectively. The EF1-α sequence HG917934 was similar to that of AY640258 (CBS164.96) and AY236901 (CMW9074) with identities of 99.7 and 99.7%, respectively. L. theobromae is a cosmopolitan fungus causing both field and storage diseases on more than 280 plant species including crops, fruits, and cash fruit trees (1,2,5). Mulberry root rot caused by L. theobromae has been reported in India (4) and ours is the first report in China. This finding clarifies the pathogen of mulberry root rot previously thought as Fusarium sp. in China, which is critical to develop management strategies to control this disease. References: (1) N. M. Celiker and T. J. Michailides. New Dis. Rep. 25:12, 2012. (2) I. H. Fischer et al. Australia Plant Dis. Notes 3:116, 2008. (3) E. Punithalingam. Botryodiplodia theobromae. CMI Descriptions of Pathogenic Fungi and Bacteria No. 519. CAB International, Wallingford, UK, 1976. (4) N. V. Radhakrishnan et al. Indian Phytopathol. 48:490, 1995. (5) B. C. Sutton. The Coelomycetes. Commonwealth Mycology Institute, Kew, Surrey, England, 1980.

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First Report of Lasiodiplodia theobromae Associated with Decline of Grapevine Rootstock Mother Plants in Spain

Plant Disease ◽

10.1094/pdis-92-5-0832b ◽

2008 ◽

Vol 92 (5) ◽

pp. 832-832 ◽

Cited By ~ 13

Author(s):

A. Aroca ◽

R. Raposo ◽

D. Gramaje ◽

J. Armengol ◽

S. Martos ◽

...

Keyword(s):

Elongation Factor ◽

Aerial Mycelium ◽

Its Region ◽

Pathogenic Fungi ◽

Vascular Lesions ◽

Malt Extract ◽

Pathogenicity Test ◽

Lasiodiplodia Theobromae ◽

First Report ◽

Mother Plants

A field of Richter 110 rootstock mother plants in Valencia Province (eastern Spain) was surveyed during November 2006 to study the mycoflora of declining plants. Two canes with stunted leaves were collected from a plant with a reduced number of shoots. No cankers or vascular lesions were observed in the collected canes. Six wood chips (1 to 2 mm thick) were taken from one basal fragment (3 to 4 cm long) of each cane, surface sterilized in 70% ethanol for 1 min, and plated on malt extract agar supplemented with 0.5 g L–1 of streptomycin sulfate. Petri dishes were incubated for 7 days at 25°C. A fungus was consistently isolated from all samples that showed the following characteristics: colonies grown on potato dextrose agar (PDA) at 25°C developed a white, aerial mycelium that turned gray after 4 to 6 days and produced pycnidia after 1 month on sterile grapevine slivers of twigs placed on the PDA surface; conidia from culture were ellipsoidal, thick walled, initially hyaline, nonseptate, and measuring 20 to 25 (22.5) × 12 to 14 (13) μm; aged conidia were brown, 1-septate with longitudinal striations in the wall; and pseudoparaphyses variable in form and length were interspersed within the fertile tissue. The fungus was identified as Lasiodiplodia theobromae (Pat.) Griffon & Maubl. from the above characteristics (2). Identity was confirmed by analysis of the nucleotide sequences of the internal transcribed spacer (ITS) region from the rRNA repeat and part of the translation elongation factor 1-alpha (EF1-α) and the β-tubulin (B-tub) genes, as done elsewhere (1,3). BLAST searches at GenBank showed a high identity with reference sequences (ITS: 100%, EF1-α: 97%; B-tub: 99%). Representative sequences of the studied DNA regions were deposited at GenBank (Accession Nos.: ITS: EU254718; EF1-α: EU254719; and B-tub: EU254720). A pathogenicity test was conducted on 1-year-old grapevine plants cv. Macabeo grafted onto Richter 110 rootstocks maintained in a greenhouse. A superficial wound was made on the bark of 10 plants with a sterilized scalpel, ≈10 cm above the graft union. A mycelial plug obtained from the margin of an actively growing fungal colony (isolate JL664) was placed in the wound and the wound was wrapped with Parafilm. Ten additional control plants were inoculated with sterile PDA plugs. All control plants grew normally, and the inoculation wound healed 3 months after inoculation. Plants inoculated with L. theobromae showed no foliar symptoms in the same period, but developed cankers variable in size surrounding the inoculation sites. Vascular necroses measuring 8.4 ± 1.5 cm (mean ± standard error) developed in the inoculated plants that were significantly longer than the controls (0.3 ± 0.2 cm). The pathogen was reisolated from all inoculated plants and no fungus was reisolated from the controls. These results confirmed the pathogenicity of L. theobromae to grapevine and points to a possible involvement of L. theobromae in the aetiology of grapevine decline as previously reported (3,4). To our knowledge, this is the first report of L. theobromae isolated from grapevine in Spain. References: (1) J. Luque et al. Mycologia 97:1111, 2005. (2) E. Punithalingam. No. 519 in: Descriptions of Pathogenic Fungi and Bacteria. CMI, Kew, Surrey, UK, 1976. (3) J. R. Úrbez-Torres et al. Plant Dis. 90:1490, 2006. (4) J. M. van Niekerk et al. Phytopathol. Mediterr. 45(suppl.):S43, 2006.

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First Report of Web Blight on Oregano (Origanum vulgare L.) Caused by Rhizoctonia solani AG-1-IB in Italy

Plant Disease ◽

10.1094/pdis-02-13-0138-pdn ◽

2013 ◽

Vol 97 (8) ◽

pp. 1119-1119 ◽

Cited By ~ 2

Author(s):

A. Garibaldi ◽

D. Bertetti ◽

P. Pensa ◽

A. Poli ◽

M. L. Gullino

Keyword(s):

Rhizoctonia Solani ◽

Its Region ◽

Morphological Characters ◽

Anastomosis Group ◽

Morphological Identification ◽

Pathogenicity Test ◽

Origanum Vulgare ◽

First Report ◽

Plastic Bags ◽

Hyphal Diameter

Origanum vulgare L., common name oregano, family Labiatae, is grown for its aromatic and medicinal properties and as ornamental. In the fall of 2012, a blight was observed in a farm located near Albenga (northern Italy) on 6% of 30,000 50-day-old plants, grown in trays in a peat/perlite mix. Semicircular, water soaked lesions appeared on leaves and stems, starting from the basal ones. As the disease progressed, blighted leaves turned brown, withered, clung to the shoots, and matted on the surrounding foliage. Eventually, infected plants died. Leaf and stem fragments taken from the margin of the diseased tissues belonging to 10 plants were disinfected for 10 s in 1% NaOCl, rinsed with sterile water, and plated on potato dextrose agar (PDA). A fungus with the morphological characters of Rhizoctonia solani was consistently recovered. Three isolates of R. solani obtained from affected plants were successfully anastomosed with R. solani isolate AG 1 (ATCC 58946). Three pairings were made for each tester strain. The hyphal diameter at the point of anastomosis was reduced, the anastomosis point was obvious, and death of adjacent cells was observed. Results were consistent with other reports on anastomosis reactions (2). Isolates from oregano were paired with R. solani isolates AG 2, 3, 4, 6, 7, or 11 and examined microscopically. Anastomosis was not observed in any of the pairings. Tests were conducted twice. Mycelium of 10-day-old isolates from oregano appeared reddish brown, coarse, and radiate. Numerous dark brown sclerotia, 0.3 to 1.0 mm diameter (average 0.7) developed within 10 days after transfer of mycelia to PDA in 90 mm diameter petri dishes at 21 to 24°C. The descriptions of mycelium and sclerotia were typical for subgroup IB Type 1 (4). The internal transcribed spacer (ITS) region of rDNA was amplified using the primers ITS1/ITS4 and sequenced. BLASTn analysis (1) of the 538 bp showed a 99% homology with the sequence of R. solani FJ746937, confirming the morphological identification of the species. The nucleotide sequence has been assigned the GenBank Accession KC493638. For pathogenicity tests, one of the isolates assigned to the anastomosis group AG-1-IB was tested by placing 9 mm diameter mycelial disks removed from PDA 10-day-old cultures of the fungus on leaves of 90-day-old oregano plants (n = 35). Thirty-five plants inoculated with non-inoculated PDA disks served as controls. Plants were covered with plastic bags and maintained in a growth chamber at 25 ± 1°C with 12 h light/dark. The first symptoms, similar to those observed in the farm, developed 3 days after inoculation. Nine days after the artificial inoculation, 50% of plants were dead. About 10 colonies of R. solani were reisolated from infected leaves of inoculated plants. Control plants remained healthy. The pathogenicity test was carried out twice with similar results. Symptoms caused by R. solani have been recently observed on O. vulgare in Greece (3). This is, to our knowledge, the first report of blight of O. vulgare caused by R. solani in Italy. References: (1) S. F. Altschul et al. Nucleic Acids Res., 25:3389, 1997. (2) D. E. Carling. Grouping in Rhizoctonia solani by hyphal anastomosis reactions. In: Rhizoctonia Species: Taxonomy, Molecular Biology, Ecology, Pathology and Disease control. Kluwer Academic Publishers, The Netherlands, pp. 37-47, 1996. (3) C. D. Holevas et al. Benaki Phytopathol. Inst., Kiphissia, Athens, 19:1-96, 2000. (4) R. T. Sherwood. Phytopathology 59:1924, 1969.

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First Report of Web Blight on Rosemary (Rosmarinus officinalis) Caused by Rhizoctonia solani AG-1-IA in Italy

Plant Disease ◽

10.1094/pdis-11-12-1012-pdn ◽

2013 ◽

Vol 97 (6) ◽

pp. 844-844 ◽

Cited By ~ 4

Author(s):

A. Garibaldi ◽

D. Bertetti ◽

P. Pensa ◽

A. Poli ◽

M. L. Gullino

Keyword(s):

Rhizoctonia Solani ◽

Ground Cover ◽

Its Region ◽

Morphological Characters ◽

Anastomosis Group ◽

Rosmarinus Officinalis ◽

Economic Losses ◽

Pathogenicity Test ◽

First Report ◽

Wheat Kernels

Rosmarinus officinalis L., family Labiatae, is an evergreen shrub used in gardens as an aromatic or ground cover plant. In the summer of 2012, a blight was observed in a farm located near Albenga (northern Italy) on 20% of 150,000 70-day-old plants, grown in trays. Water soaked lesions appeared on leaves and stems. As the disease progressed, blighted leaves turned brown, withered, clung to the shoots, and matted on the surrounding foliage. A light mycelium spread on the substrate. Disease progressed from infected plants to healthy ones and, eventually, infected plants died. Leaf and stem fragments taken from the margin of the diseased tissues belonging to 10 plants were disinfected for 10 s in 1% NaOCl, rinsed with sterile water, and plated on potato dextrose agar (PDA). A fungus with the morphological characters of Rhizoctonia solani was consistently and readily recovered. Three isolates of R. solani obtained from affected plants were successfully paired with R. solani tester strains AG 1, 2, 3, 4, 6, 7, or 11 and examined microscopically. Three pairings were made for each recovered isolate. The isolates of R. solani from rosemary anastomosed only with tester strain AG 1 (ATCC 58946). Results were consistent with other reports on anastomosis reactions (2). Tests were repeated once. Mycelium of 10-day-old isolates from rosemary appeared light brown, compact, and radiate. Numerous dark brown sclerotia, 0.7 to 2.0 mm diameter (average 1.3), developed within 10 days at 20 to 26°C. The descriptions of mycelium and sclerotia were typical for subgroup IA Type 2 (4). The internal transcribed spacer (ITS) region of rDNA was amplified using the primers ITS1/ITS4 and sequenced (GenBank Accession No. KC005724). BLASTn analysis (1) of the 657-bp showed a 99% similarity with the sequence of R. solani GU596491. For pathogenicity tests, inoculum of R. solani was prepared by growing the pathogen on wheat kernels autoclaved in 1-liter glass flasks for 8 days. One of the isolates assigned to the anastomosis group AG 1 IA was tested. Fifteen 90-day-old rosemary plants were grown in 15-liter pots in a steam disinfested peat:pomice:pine bark:clay mix (50:20:20:10) infested with 3 g/liter of infested wheat kernels, placed at the base of the stem. Fifteen plants inoculated with non-infested wheat kernels served as control treatments. Plants were covered with plastic bags and arranged in a growth chamber at 20 to 24°C with 12 h light/dark for 15 days. The first symptoms, similar to those observed in the farm, developed 10 days after inoculation. About 10 colonies of R. solani were reisolated from infected leaves and stems of each inoculated plant. Control plants remained healthy. The pathogenicity test was carried out twice with similar results. Symptoms caused by R. solani have been recently observed on R. officinalis in United States (3), India, and Brazil. This is, to our knowledge, the first report of blight of R. officinalis caused by R. solani in Italy. This disease could cause serious economic losses, because rosemary is one of the most cultivated aromatic plants in the Mediterranean region. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) D. E. Carling. Grouping in Rhizoctonia solani by hyphal anastomosis reactions. In: Rhizoctonia Species: Taxonomy, Molecular Biology, Ecology, Pathology and Disease control. Kluwer Academic Publishers, The Netherlands, 1996. (3) G. E. Holcomb. Plant Dis. 76:859, 1992. (4) R. T. Sherwood. Phytopathology 59:1924, 1969.

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First Report of Chrysoporthe deuterocubensis Causing Canker on Syzygium samarangense in Taiwan

Plant Disease ◽

10.1094/pdis-03-13-0345-pdn ◽

2013 ◽

Vol 97 (11) ◽

pp. 1508-1508

Author(s):

M. C. Fan ◽

C. C. Huang ◽

J. S. Huang ◽

S. F. Tsai ◽

H. C. Yeh ◽

...

Keyword(s):

Apple Tree ◽

Average Length ◽

Its Region ◽

Fruit Production ◽

Molecular Characteristics ◽

Malt Extract ◽

Pathogenicity Test ◽

Total Production ◽

First Report ◽

Β Tubulin

Wax apple (Syzygium samarangense Merr. & Perry, syn. Eugenia javanica Lam.) belongs to the Myrtaceae family is an important economical tree fruit in Taiwan. The total production acreage of wax apple was 5,266 ha in which more than 77% were located in Pingtung County, southern Taiwan, in 2012. Since the winter of 2010, symptoms of withering leaves and cracking branches on wax apple trees were observed in some orchards in Nanjhou and Linbian Townships, Pingtung County. Diseased trees declined gradually and resulted in reduced fruit production. On the bark of diseased twigs and branches, black conidiamata with yellowish orange conidia were usually observed. For diagnosis, tissues from symptomatic branches were excised, surface sterilized with 0.5% sodium hypochlorite, and placed on 2% water agar in petri dishes. A total of four identical fungal isolates were obtained and maintained on potato dextrose agar (PDA). To fulfill Koch's postulates, three twigs of a wax apple tree were wounded with scalpel and inoculated with each of the four isolates, one tree per isolate. A 7-day-old hyphal mat (about 7 × 18 mm) of each fungal isolate was attached on the wound, wrapped with a wet absorbent cotton and Parafilm, and then covered with a layer of aluminum foil. For the control, the twigs of a wax apple tree were inoculated with PDA plugs. The pathogenicity test was repeated once. After 30 days, withering leaves and cracking twigs were observed on inoculated twigs and the same pathogen was reisolated. Conversely, all of the non-inoculated plants remained healthy. Identification of the pathogen was conducted using its morphological, physiological, and molecular characteristics. On malt extract agar, the colony was floccose and white with hazel hues. The optimal temperature for the mycelial growth was 30°C. Conidia were hyaline, and oblong, with the average size of 4.7 ± 0.6 × 2.7 ± 0.2 μm (100 conidia). Ascostromata were semi-immersed in the bark with fusoid asci, eight ascospores per ascus. Ascospores were hyaline, 2-celled, and tapered in both ends, with the average length of 6.8 ± 0.7 × 2.4 ± 0.3 μm (100 ascospores). For molecular identification, the internal transcribed spacer (ITS) of ribosomal DNA and β-tubulin genes was amplified using the ITS1/ITS4 (3), Bt1a/Bt1b, and Bt2a/Bt2b (1) primer pairs. The gene sequences were deposited in GenBank (Accessions KC792616, KC792617, KC792618, and KC792619 for the ITS region; KC792620, KC792621, KC792622, and KC792623 for Bt1 region, and KC812732, KC812733, KC812734, and KC812735 for Bt2 region) and showed 99 to 100% identity to the Chrysoporthe deuterocubensis isolate CMW12745 (DQ368764 for ITS region; GQ290183 for Bt1 region, and DQ368781 for Bt2 region). In addition, the Bt1 region of the β-tubulin gene consisted of two restriction sites for AvaI and one restriction site for HindIII. This is identical to the description of C. deuterocubensis, a cryptic species in C. cubensis, by Van Der Merwe et al. (2). According to these results, the pathogen was identified as C. deuterocubensis Gryzenh. & M. J. Wingf. To the best of our knowledge, this is the first report of canker disease caused by C. deuterocubensis on S. samarangense in Taiwan. References: (1) N. L. Glass and G. C. Donaldson. Appl. Environ. Microbiol. 61:1323, 1995. (2) N. A. Van Der Merwe et al. Fungal Biol. 114:966, 2010. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

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First Report of Gray Mold Caused by Botrytis cinerea on Stevia rebaudiana in Italy

Plant Disease ◽

10.1094/pdis-93-3-0318a ◽

2009 ◽

Vol 93 (3) ◽

pp. 318-318

Author(s):

A. Garibaldi ◽

D. Bertetti ◽

P. Pensa ◽

M. L. Gullino

Keyword(s):

Botrytis Cinerea ◽

Stevia Rebaudiana ◽

Its Region ◽

Morphological Characters ◽

Gray Mold ◽

Fluorescent Light ◽

Pathogenicity Test ◽

Leaf Extracts ◽

First Report ◽

Potted Plants

Stevia rebaudiana (sweetleaf) is a perennial shrub belonging to the Asteraceae family and is widely grown for its sweet leaves. With its extracts having as much as 300 times the sweetness of sugar, this species is used in many countries for the production of sugar substitutes. However, in Italy, as well as in other countries, this species cannot be grown for the use of its leaf extracts. This plant is grown in a few nurseries in the Albenga Region (northern Italy) as potted plants. In February of 2008, 3-month-old plants grown in plastic pots (14-cm diameter) under glasshouse on heated benches started showing symptoms of a previously unknown blight. The temperature in the glasshouse ranged between 16 and 20°C and plants were watered by sprinkle irrigation. Leaves, starting from the basal ones, showed small, brown spots that spread across the entire leaf surface. Subsequently, the crown and stem were infected, and the pathogen developed abundant, soft, gray mycelium on leaves and stems and in the middle of the heads of S. rebaudiana. Flowers were not present when the symptoms appeared. Severely infected leaves dried out and became necrotic. The disease was observed in one nursery in which 5% of the plants were affected. The margins of the lesions were excised from leaves, immersed in a solution containing 1% sodium hypochlorite, and then cultured on potato dextrose agar (PDA) medium. A fungus produced abundant mycelium when incubated under constant fluorescent light at 22 ± 1°C after 10 days. The conidia were smooth, hyaline, ovoid, measuring 15.5 to 8.3 × 11.1 to 7.3 (average 11.6 × 8.6) μm, and were similar to those described for Botrytis cinerea. Conidiophores were slender and branched with enlarged apical cells bearing conidia on short sterigmata. The identity of the fungus was also confirmed by the production of numerous, small, black sclerotia on PDA plates incubated for 20 days at 8 ± 1°C. Sclerotia were dark and irregular with a diameter ranging from 1 to 2 mm. These morphological characters identified the fungus as B. cinerea (2). The internal transcribed spacer (ITS) region of rDNA was amplified using primers ITS4/ITS6 and sequenced. BLAST analysis (1) of the 780-bp segment showed a 100% homology with the sequence of Botryotinia fuckeliana (perfect stage of B. cinerea). The nucleotide sequence has been assigned GenBank Accession No. FJ486270. Pathogenicity tests were performed by spraying leaves of six healthy 6-month-old potted S. rebaudiana plants with a 105 conidia/ml suspension. Six plants sprayed with water only served as controls. Plants were covered with plastic bags for 3 days after inoculation to maintain high relative humidity and were placed in a growth chamber at 20 ± 1°C. The first foliar lesions developed on leaves 4 days after inoculation, whereas control plants remained healthy. B. cinerea was consistently reisolated from these lesions. The pathogenicity test was completed twice. To our knowledge, this is the first report of the presence of B. cinerea on S. rebaudiana in Italy. The disease has been reported in Ukraine (3) and more recently in Japan (4). The economic importance of this disease is at the moment limited. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) H. L. Barnett and B. B. Hunter. Illustrated Genera of Imperfect Fungi. Burgess Publishing Company, Minneapolis, MN, 1972. (3) J. Takeuch and H. Horie. Annu. Rep. Kanto-Tosan Plant Prot. Soc. 53:87, 2006. (4) V. F. Zubenko et al. Zash. Rast. 18, 1991.

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First Report of Lasiodiplodia theobromae Causing Postharvest Fruit Rot on Guava (Psidium guajava) in Malaysia

Plant Disease ◽

10.1094/pdis-12-20-2732-pdn ◽

2021 ◽

Author(s):

Kar Yan Zee ◽

Norhayu Asib ◽

Siti Izera Ismail

Keyword(s):

Morphological Characteristics ◽

Its Region ◽

Psidium Guajava ◽

Fruit Rot ◽

Pathogenicity Test ◽

Tropical Fruit ◽

Lasiodiplodia Theobromae ◽

First Report ◽

Guava Fruit ◽

Initial Symptoms

Guava (Psidium guajava L.) is an economically important tropical fruit crop and is cultivated extensively in Malaysia. In September and October 2019, postharvest fruit rot symptoms were observed on 30% to 40% of guava fruit cv. Kampuchea in fruit markets of Puchong and Ipoh cities in the states of Selangor and Perak, Malaysia. Initial symptoms appeared as brown, irregular, water-soaked lesions on the upper portion of the fruit where it was attached to the peduncle. Subsequently, lesions then progressed to cover the whole fruit (Fig.1A). Lesions were covered with an abundance of black pycnidia and grayish mycelium. Ten symptomatic guava fruit were randomly collected from two local markets for our investigation. For fungal isolation, small fragments (5×5 mm) were excised from the lesion margin, surface sterilized with 0.5% NaOCl for 2 min, rinsed three times with sterile distilled water, placed on potato dextrose agar (PDA) and incubated at 25 °C with 12-h photoperiod for 2-3 days. Eight single-spore isolates with similar morphological characteristics were obtained and two representative isolates (P8 and S9) were characterized in depth. Colonies on PDA were initially composed of grayish-white aerial mycelium, but turned dark-gray after 7 days (Fig. 1B). Abundant black pycnidia were observed after incubation for 4 weeks. Immature conidia were hyaline, aseptate, ellipsoid, thick-walled, and mature conidia becoming dark brown and 1-septate with longitudinal striations, 25.0 − 27.0 ± 2.5 × 13.0 − 14.0 ± 1.0 μm (n = 30) (Fig.1C, D). On the basis of morphology, both representative isolates were identified as Lasiodiplodia theobromae (Pat.) Griffon & Maubl. (Alves et al. 2008). For molecular identification, genomic DNA of the two isolates was extracted using the DNeasy plant mini kit (Qiagen, USA). The internal transcribed spacer (ITS) region of rDNA and translation elongation factor 1-alpha (EF1-α) genes were amplified using ITS5/ITS4 and EF1-728F/EF1-986R primer set, respectively (White et al. 1990, Carbone and Kohn 1999). BLASTn analysis of the resulting ITS and EF1-α sequences indicated 100% identity to L. theobromae ex-type strain CBS 164.96 (GenBank accession nos: AY640255 and AY640258, respectively) (Phillips et al. 2013). The ITS (MW380428, MW380429) and EF1-α (MW387153, MW387154) sequences were deposited in GenBank. Phylogenetic analysis using the maximum likelihood based on the combined ITS-TEF sequences indicated that the isolates formed a strongly supported clade (100% bootstrap value) to the related L. theobromae (Kumar et al. 2016) (Fig.2). A pathogenicity test of two isolates was conducted on six healthy detached guava fruits per isolate. The fruit were surface sterilized using 70% ethanol and rinsed twice with sterile water prior inoculation. The fruit were wound-inoculated using a sterile needle according to the method of de Oliveira et al. (2014) and five-mm-diameter mycelial agar plugs from 7-days-old PDA culture of the isolates were placed onto the wounds. Six additional fruit were wound inoculated using sterile 5-mm-diameter PDA agar plugs to serve as controls. Inoculated fruit were placed in sterilized plastic container and incubated in a growth chamber at 25 ± 1 °C, 90% relative humidity with a photoperiod of 12-h. The experiment was conducted twice. Five days after inoculation, symptoms as described above developed on the inoculated sites and caused a fruit rot, while control treatment remained asymptomatic. L. theobromae was reisolated from all symptomatic tissues and confirmed by morphological characteristics and confirmed by PCR using ITS region. L. theobromae has recently been reported to cause fruit rot on rockmelon in Thailand (Suwannarach et al. 2020). To our knowledge, this is the first report of L. theobromae causing postharvest fruit rot on guava in Malaysia. The occurrence of this disease needs to be monitored as this disease can reduce the marketable yield of guava. Preventive strategies need to be developed in the field to reduce postharvest losses.

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First Report of Collar and Root Rot Caused by Pythium ultimum on Coriander in Italy

Plant Disease ◽

10.1094/pdis-94-9-1167b ◽

2010 ◽

Vol 94 (9) ◽

pp. 1167-1167 ◽

Cited By ~ 3

Author(s):

A. Garibaldi ◽

G. Gilardi ◽

M. L. Gullino

Keyword(s):

Root Rot ◽

Its Region ◽

Pythium Ultimum ◽

Morphological Characters ◽

Fluorescent Light ◽

Academic Press ◽

Medicinal Uses ◽

First Report ◽

Young Leaves ◽

Collar And Root Rot

Coriander, Coriandrum sativum L., is an annual herb in the family Apiaceae. This plant, native to southern Europe, northern Africa, and southeastern Asia, is used in cooking as well as for medicinal uses. The leaves are commonly referred to as cilantro. In October 2009, severe outbreaks of a previously unknown root rot were observed in a commercial field located in the Alessandria Province (northern Italy) on 20-day-old plants belonging to cv. Comune. Five percent of plants were affected, showing stunting and extensive chlorosis starting on external leaves that eventually wilted. Root systems and collars of diseased plants appeared rotted. In advanced stages, young leaves were affected and the plants eventually collapsed and tissues dried out. Tissue fragments of 1 mm2 were excised from the roots of infected plants, dipped in a solution containing 1% sodium hypochlorite, and plated on potato dextrose agar (PDA) and a medium selective for Oomycetes (3). Plates were incubated under constant fluorescent light at 22 ± 1°C for 4 to 5 days. One isolate, grown on V8 medium (vegetable mix, 300 g; agar, 15 g; CaCO3, 1.5g; and distilled water, 1 liter), and observed under a light microscope showed hyphae generally aseptate, ranging from 1.3 to 6.24 μm in diameter, and produced sporangia consisting of complexes of swollen hyphal branches. Oogonia were globose, smooth, and 20.3 to 33.4 (average 25.4) μm in diameter. Antheridia were monoclinous, extending from immediately below oogonium, and measured 10.8 to 17.8 × 7.6 to 12.7 (average 14.4 × 10.4) μm. Oospores were single, globose, aplerotic and thick walled, and 15.8 to 24.2 (average 17.8) μm in diameter. These morphological characters were used to identify the microorganism as a Pythium sp. (3). The internal transcribed spacer (ITS) region of rDNA of a single isolate was amplified using the primers ITS1/ITS4 (2,4) and sequenced. BLAST analysis (1) of the 874-bp segment showed a 100% homology with the sequence of Pythium ultimum. The nucleotide sequence has been assigned the GenBank Accession No. GU478314. Pathogenicity tests were performed twice on coriander cv. Comune. Plants were grown in 2-liter pots containing a Brill Type 5 substrate (Brill Substrate GmbH & Co. KG, Niedersachsen, Germany) consisting of 15% blond peat, 85% black peat, pH 5.5 to 6, and pasteurized at 80°C for 30 min. The potting mix was infested at a rate of 5 g/liter with wheat and hemp kernels colonized with one strain of P. ultimum. Ten plants (1 plant per pot) were grown in the infested substrate and 10 plants were grown in noninfested substrate. Plants were kept in a growth chamber at 20°C. The first symptoms, consisting of reduced growth and chlorosis, developed within 7 days, while control plants remained healthy. P. ultimum was consistently isolated from the roots. To our knowledge, this is the first report of P. ultimum causing disease of coriander in Italy as well as worldwide. At this time, the economic importance of Pythium rot on coriander in Italy remains unknown. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) W. Chen et al. Exp. Mycol.16:22, 1992. (3) T. Watanabe. Pictorial Atlas of Soil and Seed Fungi. CRC Press, Boca Raton, FL, 2002. (4) T. J. White et al. Page 38 in: PCR protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, 1990.

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First Report of Leaf Blight on Woodland Sage Caused by Rhizoctonia solani AG 1 in Italy

Plant Disease ◽

10.1094/pdis-94-8-1071c ◽

2010 ◽

Vol 94 (8) ◽

pp. 1071-1071 ◽

Cited By ~ 2

Author(s):

A. Garibaldi ◽

G. Gilardi ◽

D. Bertetti ◽

M. L. Gullino

Keyword(s):

Rhizoctonia Solani ◽

Aqueous Suspension ◽

Its Region ◽

Morphological Characters ◽

Leaf Blight ◽

Pathogenicity Test ◽

First Report ◽

Leaf Petiole ◽

Perennial Plant ◽

Potted Plants

Woodland sage (Salvia nemorosa L.; Lamiaceae) is a hardy herbaceous perennial plant that is easy to grow and propagate and is used in parks and grown as potted plants. During the summer of 2009 in a nursery near Torino in northern Italy, a leaf blight was observed on 30-day-old plants of cv. Blau Koenigin grown in pots under shade. Semicircular, water-soaked lesions developed on leaves just above the soil line at the leaf-petiole junction and later along leaf margins. Lesions expanded along the midvein until the entire leaf was destroyed. Blighted leaves turned brown, withered, and clung to the shoots. No symptoms were observed on the roots. Severely infected plants died. Diseased tissue was disinfested for 10 s in 1% NaOCl, rinsed with sterile water, and plated on potato dextrose agar (PDA) amended with 25 mg/liter of streptomycin sulfate. A fungus with morphological characters of Rhizoctonia solani (3) was consistently recovered. Ten-day-old mycelium grown on PDA at 22 ± 1°C appeared light brown, rather compact, and with radial growth. Sclerotia were irregular and measured between 0.5 and 2 mm. Pairings were made with tester isolates of AG 1, 2, 3, 4, 5, 6, 7, 11, and AG B1. The only successful anastomosis was with tester isolate AG 1 (ATCC 58946). The hyphal diameter at the point of anastomosis was reduced, the anastomosis point was obvious, and cell death of adjacent cells was observed. Results were consistent with other reports on anastomosis reactions (2). The description of sclerotia of the isolate AG1 was typical for subgroup 1A Type 2 (3). The internal transcribed spacer (ITS) region of rDNA was amplified using primers ITS4/ITS6 and sequenced. BLASTn analysis (1) of the 688 bp showed a 100% homology with the sequence of R. solani AG-1A and the nucleotide sequence has been assigned (GenBank Accession No. HM044764). For pathogenicity tests, the inoculum of one isolate of R. solani from the nursery was prepared by growing the pathogen on PDA for 7 days. The foliage of 30-day-old potted plants of S. nemorosa cv. Blau Koenigin was artificially inoculated with an aqueous suspension of PDA and mycelium fragments (1 g per mycelium per plant) prepared from cultures with a blender. Plants were covered with plastic bags for 3 days. Plants inoculated with water and PDA fragments alone served as control treatments. Plants were maintained in a glasshouse at 20 to 25°C. The first symptoms, similar to those observed in the nursery, developed 7 days after foliar inoculation. R. solani was consistently reisolated from infected leaves. Control plants remained healthy. The pathogenicity test was carried out twice with similar results. To our knowledge, this is the first report of leaf blight of S. nemorosa caused by R. solani in Italy as well as worldwide. The importance of the disease is still unknown. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) D. E. Carling. Page 35 in: Rhizoctonia Species: Taxonomy, Molecular Biology, Ecology, Pathology and Disease Control. Kluwer Academic Publishers, the Netherlands, 1996. (3) B. Sneh et al. Identification of Rhizoctonia Species. The American Phytopathological Society, St Paul, MN, 1991.

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First report of postharvest fruit rot on passion fruit (Passiflora edulis) caused by Lasiodiplodia theobromae in Mainland China

Plant Disease ◽

10.1094/pdis-06-20-1346-pdn ◽

2020 ◽

Author(s):

Wu Zhang ◽

Xue Li Niu ◽

Jin Yu Yang

Keyword(s):

Southern China ◽

Morphological Characteristics ◽

Its Region ◽

Passion Fruit ◽

Fruit Rot ◽

Pathogenicity Test ◽

Single Spore ◽

Passiflora Edulis ◽

Lasiodiplodia Theobromae ◽

First Report

As an economically important tropical and subtropical fruit crop, passion fruit (Passiflora edulis Sims) is widely planted in many provinces of southern China. In April 2017, postharvest fruit rot was observed on 15% to 25% of passion fruit in several fruit markets of Zhanjiang City in Guangdong Province. Initial disease symptoms on infected fruit were irregular, brown, water-soaked lesions, which enlarged into large black and sunken patches. Lesions were usually covered with an abundance of little black dots (pycnidia) and black-gray hyphae. For the pathogen isolation, fifteen symptomatic fruit were randomly collected from three local markets. Fourteen single-spore fungal isolates with similar morphology ware isolated from the infected tissues. Two isolates (ZW 49-1 and ZW 50-1) were randomly selected to further study. The colonies on PDA were initially greyish-white and became dark-gray with age. Abundant globular and irregular pycnidia were observed after incubation at 25 °C for 3 weeks. The conidia of the fungus were initially hyaline, unicellular, apex rounded, thick-walled, and ellipsoid, becoming dark brown, bicellular with longitudinal striations at maturity, 26.4 ± 2.5 × 13.4 ± 1.2 μm (n = 50). The morphology of the fungus resembled Lasiodiplodia theobromae (Pat.) Griff. & Maubl. (Phillips et al. 2013). To confirm species identification, the partial internal transcribed spacer (ITS) region of rDNA, translation elongation factor-alpha (EF1-α) and β-tubulin (TUB) gene were amplified from genomic DNA of the two isolates with the ITS1/ITS4, EF1-688F/EF1-986R, and Bt2a/Bt2b primers, respectively (Glass and Donaldson 1995; Alves et al. 2008; White et al. 1990). Base on the BLASTn analysis, the ITS (MT644473, MT644474), EF1-α (MT649210, MT649211) and TUB (MT649212, MT649213) sequences of both isolates were 100%, 99% and 100% similarity to the L. theobromae CBS 164.96 ex-type sequences in the NCBI database (AY640255, AY640258, and KU887532, respectively) (Phillips et al. 2013). For pathogenicity test, asymptomatic passion fruit were previously disinfested in 0.5 % sodium hypochlorite and superficially wounded with a sterile needle. Five-mm-diameter plugs with mycelial taken from 5-day-old PDA colonies were placed on the wounds. Sterile PDA plugs were used as negative controls. Each treatment had five replicates and the test was repeated twice. Fruit were maintained in plastic boxes to keep at 25°C for one week. One week after inoculation, gray mycelia had covered a majority of the fruit surface and caused a black, sunken rot. The inoculated fungus was reisolated and confirmed as L. theobromae by morphological characteristics. The mock inoculated fruit remained asymptomatic. The occurrence of fruit rot on passion fruit caused by L. theobromae was reported in Taiwan, China recently (Huang et al., 2019). To our knowledge, this is the first report of L. theobromae causing postharvest fruit rot on passion fruit in the Chinese mainland.

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First report of Mycoleptodiscus terrestris causing root rot of soybean in Indiana

Plant Disease ◽

10.1094/pdis-09-20-2023-pdn ◽

2020 ◽

Author(s):

Christopher Detranaltes ◽

Guohong Cai

Keyword(s):

Root Rot ◽

Biological Control Agent ◽

Lateral Roots ◽

Its Region ◽

Selective Medium ◽

Internal Transcribed Spacers ◽

Root Tissue ◽

Pathogenicity Test ◽

First Report ◽

Soybean Seedlings

During the summers in 2019 and 2020, 137 soybean (Glycine max (L.) Merr) seedlings (V1-V3 stage) showing stunting, delayed emergence, and/or crown lesions were collected at Purdue’s Agronomy Center for Research and Education in West Lafayette, Indiana. Four seedlings were stunted with reddish-brown girdled lesions along the hypocotyl and crown, rotted tap and lateral roots, and brown discoloration of the cortex and vascular tissues. Four fungal isolates (AC4, AC58, AC96, and AC127) were recovered by plating surface-sterilized symptomatic root tissue onto water agar plates and incubating on the benchtop until mycelia emerged. The growing hyphal tips were transferred to the semi-selective medium DCPA (Andrews and Pitt 1986). On potato dextrose agar, the fungal colonies developed olivaceous green mycelia which melanized into a mat of black microsclerotia with time and no conidia were observed. On 1.5% water agar plates amended with twice autoclaved soybean leaf and root tissue collected from flowering soybean plants, conidia were formed in sporodochia in darkness at 28 οC within one week. Conidia were 1-2 septate, cylindrical with two setae on either end, and measured 20.8 to 26.4 x 4 to 5.6 μm (average 23.9 x 4.7 μm, n=20). The morphological characters matched with the description of Mycoleptodiscus terrestris (Gerd.) Ostaz (Gerdemann 1953). Species identification was further confirmed by sequencing the internal transcribed spacers (ITS) region of rDNA amplified by ITS1 and ITS4 primers (White et al. 1990) and the translation elongation factor 1 alpha (TEF1-α) gene using 983F and 1567R primers with annealing temperature at 53 ○C (Rehner and Buckley 2005). The sequences were deposited in GenBank under the following accession numbers: ITS: MW002684, MT998441, MW010258, and MW010260; and TEF1-α: MW015941-MW015944. The GenBank BLAST searches revealed 100% identity in the ITS region (accession NR_145373.1) and 99.75% identity in the TEF1-α region (MK495977.1) to M. terrestris. Pathogenicity test was conducted on soybean seedlings (cv. Williams) at V1 growth stage using a root dip assay. Isolate AC58 was grown in a modified cotton seed meal broth (CSMB) to produce microsclerotia as inoculum (Gray 1978; Shearer and Jackson 2006). Microsclerotia concentration was measured using a hemocytometer and adjusted to 1.5 x 104 per ml. Five soybean seedlings each were dipped into inoculum or sterile CSMB for 30 minutes then planted individually in vermiculite-filled Styrofoam cups placed on flooded trays in 16-hr photoperiod light racks at room temperature. Seven days after inoculation, all inoculated plants were visibly stunted with root and crown symptoms identical to field symptoms while all controls were healthy. M. terrestris was successfully re-isolated from inoculated plants, but not from the controls, and identified by morphology and sequencing as above. M. terrestris has been previously reported causing root rot of soybean in Illinois (Gray 1978) and Wisconsin (Smith et al. 1998). To our knowledge, this is the first report of M. terrestris infecting soybean in Indiana. Increased geographic distribution of this pathogen warrants more attention for its control. M. terrestris has been proposed as a biological control agent against multiple aquatic weeds (Verma and Charudattan 1993; Shearer and Jackson 2006). Introduction of this fungus into soybean production regions should be avoided.

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