First Report of Black Stem of Avicennia marina Caused by Fusarium equiseti in China

Avicennia marina is a pioneer species of mangroves, a woody plant community that periodically emerges in the intertidal zone of estuarine regions in tropical and subtropical regions. In February 2013, a new disease that caused the stems of A. marina to blacken and die was found in Techeng Island of Zhanjiang, Guangdong Province, China. Initial symptoms of the disease were water-soaked brown spots on the biennial stems that coalesced so whole stems browned, twigs and branches withered, leaves defoliated, and finally trees died. This disease has the potential to threaten the ecology of the local A. marina community. From February to May 2013, 11 symptomatic trees were collected in three locations on the island and the pathogen was isolated as followed: tissues were surface disinfected with 75% ethanol solution (v/v) for 20 s, soaked in 0.1% mercuric chloride solution for 45 s, rinsed with sterilized water three times, dried, placed on potato dextrose agar (PDA), and incubated for 3 to 5 days at 28°C without light. Five isolates (KW1 to KW5) with different morphological characteristics were obtained, and pathogenic tests were done according Koch's postulates. Fresh wounds were made with a sterile needle on healthy biennial stems of A. marina, and mycelial plugs of each isolate were applied and covered with a piece of wet cotton to maintain moisture. All treated plants were incubated at room temperature. Similar symptoms of black stem were observed only on the stems inoculated the isolate KW5 after 35 days, while the control and all stems inoculated with the other isolates remained symptomless. An isolate similar to KW5 was re-isolated from the affected materials. The pathogenic test was repeated three times with the same conditions and it was confirmed that KW5 was the pathogen causing the black stem of A. marina. Hyphal tips of KW5 were transferred to PDA medium in petri dishes for morphological observation. After 48 to 72 h, white, orange, or brown flocculence patches of KW5 mycelium, 5.0 to 6.0 cm in diameter, grew. Tapering and spindle falciform macroconidia (11 to 17.3 μm long × 1.5 to 2.5 μm wide) with an obviously swelled central cell and narrow strips of apical cells and distinctive foot cells were visible under the optical microscope. The conidiogenous cells were intertwined with mycelia and the chlamydospores were globose and formed in clusters. These morphological characteristics of the isolate KW5 are characteristic of Fusarium equiseti (1). For molecular identification, the ITS of ribosomal DNA, β-tubulin, and EF-1α genes were amplified using the ITS4/ITS5 (5), T1/T2 (2), and EF1/EF2 (3) primer pairs. These sequences were deposited in GenBank (KF515650 for the ITS region; KF747330 for β-tubulin region, and KF747331 for EF-1α region) and showed 98 to 99% identity to F. equiseti strains (HQ332532 for ITS region, JX241676 for β-tubulin gene, and GQ505666 for EF-1α region). According to both morphological and sequences analysis, the pathogen of the black stem of A. marina was identified as F. equiseti. Similar symptoms on absorbing rootlets and trunks of A. marina had been reported in central coastal Queensland, but the pathogen was identified as Phytophthora sp. (4). Therefore, the disease reported in this paper differs from that reported in central coastal Queensland. To our knowledge, this is the first report of black stems of A. marina caused by F. equiseti in China. References: (1) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual, 1st ed. Wiley-Blackwell, Hoboken, NJ, 2006. (2) K. O'Donnell and E. Cigelnik. Mol. Phylogenet. Evol. 7:103, 1997. (3) K. O'Donnell et al. Proc. Natl. Acad. Sci. USA. 95:2044, 1998. (4) K. G. Pegg. Aust et al. Plant Pathol. 3:6, 1980. (5) A. W. Zhang et al. Plant Dis. 81:1143, 1997.

Download Full-text

First Report of Nigrospora Leaf Blight on Tea Caused by Nigrospora sphaerica in India

Plant Disease ◽

10.1094/pdis-05-14-0545-pdn ◽

2015 ◽

Vol 99 (3) ◽

pp. 417-417 ◽

Cited By ~ 11

Author(s):

J. Dutta ◽

S. Gupta ◽

D. Thakur ◽

P. J. Handique

Keyword(s):

Camellia Sinensis ◽

West Bengal ◽

Morphological Characteristics ◽

Its Region ◽

Causative Organism ◽

Distilled Water ◽

Conidial Suspension ◽

Tea Leaves ◽

First Report ◽

Initial Symptoms

Tea [Camellia sinensis (L.) O. Kuntze] is an economically important non-alcoholic caffeine-containing beverage crop widely cultivated for leaves in India, especially in the Darjeeling district of West Bengal. In May 2012, distinct blight symptoms were observed on leaves of popular tea cultivars AV-2, Tukdah 78, Rungli Rungliot 17/144, and Bannockburn 157 in commercial tea estates of the Darjeeling district. This disease reduces yield and quality of the leaves. The initial symptoms were frequently observed on the young leaf margins and apices. Foliar symptoms are characterized by grayish to brown, semicircular or irregular shaped lesions, often surrounded by pale yellow zones up to 9 mm in diameter. The lesions later expand and the affected leaves turn grayish to dark brown and eventually the dried tissue falls, leading to complete defoliation of the plant. The disease causes damage to leaves of all ages and is severe in young leaves. A portion of the symptomatic leaf tissues were surface sterilized in 70% ethanol for 30 s, then in 2% NaClO for 3 min, rinsed three times in sterile distilled water, and plated onto potato dextrose agar (PDA). The fungal colonies were initially white and then became grayish to brown with sporulation. Conidia were spherical to sub spherical, single-celled, black, 19 to 21 μm in diameter, and were borne on a hyaline vesicle at the tip of each conidiophore. Morphological characteristics of the isolates were concurring to those of Nigrospora sphaerica (1). Moreover, the internal transcribed spacer (ITS) region of the ribosomal RNA was amplified by using primers ITS1 and ITS4 and sequenced (GenBank Accession No. KJ767520). The sequence was compared to the GenBank database through nucleotide BLAST search and the isolate showed 100% similarity to N. sphaerica (KC519729.1). On the basis of morphological characteristics and nucleotide homology, the isolate was identified as N. sphaerica. Koch's postulates were fulfilled in the laboratory on tea leaves inoculated with N. sphaerica conidial suspension (106 conidia ml−1) collected from a 7-day-old culture on PDA. Six inoculated 8-month-old seedlings of tea cultivars AV-2 and S.3/3 were incubated in a controlled environment chamber at 25°C and 80 to 85% humidity with a 12-h photoperiod. In addition, three plants of each cultivar were sprayed with sterile distilled water to serve as controls. Twelve to 14 days after inoculation, inoculated leaves developed blight symptoms similar to those observed on naturally infected tea leaves in the field. No symptoms were observed on the control leaves. The pathogen was re-isolated from lesions and its identity was confirmed by morphological characteristics. It was reported that N. sphaerica is frequently encountered as a secondary invader or as a saprophyte on many plant species and also as a causative organism of foliar disease on several hosts worldwide (2,3). To our knowledge, this is first report of N. sphaerica as a foliar pathogen of Camellia sinensis in Darjeeling, West Bengal, India, or worldwide. References: (1) M. B. Ellis. Dematiaceous Hyphomycetes. CMI, Kew, Surrey, UK, 1971. (2) D. F. Farr and A. Y. Rossman. Fungal Databases, Syst. Mycol. Microbiol. Lab., ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ July 01, 2013. (3) E. R. Wright et al. Plant Dis. 92:171, 2008.

Download Full-text

First report of Nigrospora sphaerica causing fruit dried-shrink disease in Akebia trifoliata from China

Plant Disease ◽

10.1094/pdis-11-20-2471-pdn ◽

2021 ◽

Author(s):

Xiujing Hong ◽

Shijia Chen ◽

linchao Wang ◽

Bo Liu ◽

Yuruo Yang ◽

...

Keyword(s):

Elongation Factor ◽

Morphological Characteristics ◽

Its Region ◽

Average Diameter ◽

Ethanol Solution ◽

Likelihood Method ◽

Conidial Suspension ◽

Multiple Sequence ◽

First Report ◽

Pure Cultures

Akebia trifoliata, a recently domesticated horticultural crop, produces delicious fruits containing multiple nutritional metabolites and has been widely used as medicinal herb in China. In June 2020, symptoms of dried-shrink disease were first observed on fruits of A. trifoliata grown in Zhangjiajie, China (110.2°E, 29.4°N) with an incidence about 10%. The infected fruits were shrunken, colored in dark brown, and withered to death (Figure S1A, B). The symptomatic fruits tissues (6 × 6 mm) were excised from three individual plants, surface-disinfested in 1% NaOCl for 30s and 70% ethanol solution for 45s, washed, dried, and plated on potato dextrose agar (PDA) containing 50 mg/L streptomycin sulfate in the dark, and incubated at 25℃ for 3 days. Subsequently, hyphal tips were transferred to PDA to obtain pure cultures. After 7 days, five pure cultures were obtained, including two identical to previously reported Colletotrichum gloeosporioides causing leaf anthracnose in A. trifoliata (Pan et al. 2020) and three unknown isolates (ZJJ-C1-1, ZJJ-C1-2, and ZJJ-C1-3). The mycelia of ZJJ-C1-1, ZJJ-C1-2 and ZJJ-C1-3 were white, and formed colonies of approximate 70 mm (diameter) in size at 25℃ after 7 days on potato sucrose agar (PSA) plates (Figure S1C). After 25 days, conidia were formed, solitary, globose, black, shiny, smooth, and 16-21 μm in size (average diameter = 18.22 ± 1.00 μm, n = 20) (Figure S1D). These morphological characteristics were similar to those of N. sphaerica previously reported (Li et al. 2018). To identify species of ZJJ-C1-1, ZJJ-C1-2 and ZJJ-C1-3, the internal transcribed spacer (ITS) region, β-tubulin (TUB2), and the translation elongation factor 1-alpha (TEF1-α) were amplified using primer pairs including ITS1/ITS4 (Vilgalys and Hester 1990), Bt-2a/Bt-2b (Glass and Donaldson 1995), and EF1-728F/EF-2 (Zhou et al. 2015), respectively. Multiple sequence analyses showed no nucleotide difference was detected among genes tested except ITS that placed three isolates into two groups (Figure S2). BLAST analyses determined that ZJJ-C1-1, ZJJ-C1-2 and ZJJ-C1-3 had 99.73% to N. sphaerica strains LC2705 (KY019479), 100% to LC7294 (KY019397), and 99.79-100% to LC7294 (KX985932) or LC7294 (KX985932) based on sequences of TUB2 (MW252168, MW269660, MW269661), TEF-1α (MW252169, MW269662, MW269663), and ITS (MW250235, MW250236, MW192897), respectively. These indicated three isolates belong to the same species of N. sphaerica. Based on a combined dataset of ITS, TUB2 and TEF-1α sequences, a phylogenetic tree was constructed using Maximum likelihood method through IQ-TREE (Minh et al. 2020) and confirmed that three isolates were N. sphaerica (Figure S2). Further, pathogenicity tests were performed. Briefly, healthy unwounded fruits were surface-disinfected in 0.1% NaOCl for 30s, washed, dried and needling-wounded. Then, three fruits were inoculated with 10 μl of conidial suspension (1 × 106 conidia/ml) derived from three individual isolates, with another three fruits sprayed with 10 μl sterilized water as control. The treated fruits were incubated at 25℃ in 90% humidity. After 15 days, all the three fruits inoculated with conidia displayed typical dried-shrink symptoms as those observed in the farm field (Figure S1E). The decayed tissues with mycelium and spores could be observed on the skin or vertical split of the infected fruits after 15 days’ inoculation (Figure S1F-H). Comparably, in the three control fruits, there were no dried-shrink-related symptoms displayed. The experiment was repeated twice. The re-isolated pathogens were identical to N. sphaerica determined by sequencing the ITS, TUB2 and TEF-1α. Previous reports showed N. sphaerica could cause postharvest rot disease in kiwifruits (Li et al. 2018). To our knowledge, this is the first report of N. sphaerica causing fruits dried-shrink disease in A. trifoliata in China.

Download Full-text

First Report of Phytophthora syringae on Cedrus libani in Turkey

Plant Disease ◽

10.1094/pdis-09-13-0962-pdn ◽

2014 ◽

Vol 98 (6) ◽

pp. 846-846 ◽

Cited By ~ 1

Author(s):

T. Doğmuş-Lehtijärvi ◽

A. G. Aday Kaya ◽

A. Lehtijärvi ◽

T. Jung

Keyword(s):

Average Length ◽

Morphological Characteristics ◽

Soil Samples ◽

Morphological Identification ◽

First Report ◽

Cedrus Libani ◽

Initial Symptoms ◽

Lebanon Cedar ◽

Forest Nurseries ◽

Β Tubulin

Cedrus libani, commonly known as Lebanon cedar, is one of the most important coniferous tree species in Turkey. Its main distribution is in the Taurus Mountains in the Mediterranean Region. The total area of pure Taurus cedar forest covers 109,440 ha in Turkey, all located in the southwestern regions of the country. Due to its drought resistance, Taurus cedar has been commonly used for afforestations in these semi-arid areas (1). In September 2011, during surveys for Phytophthora spp. in forest nurseries in Adapazari and İzmir in eastern Turkey, initial symptoms such as death of fine roots, yellowing, and wilting of Taurus cedar seedlings were observed. Soil samples were collected from 10 symptomatic C. libani seedlings and isolation tests for Phytophthora species were carried out using leaflets from young Quercus suber, Azalea sp., and Rhodendron sp. saplings as baits floated over flooded soil. Necrotic baits were blotted dry, cut into small pieces, and placed on selective PARPNH carrot agar. Out growing colonies were subcultured on carrot agar and kept at 12°C for morphological and molecular identifications (2). In total, six Pythiaceous isolates were obtained from the C. libani soil samples. The isolates were investigated using a light microscope and grouped according to their morphological characteristics (3). DNA was extracted from two representative isolates using Qiagen DNeasy Plant Mini Kit following the manufacturer's instructions. PCR amplifications and sequencing of the internal transcribed spacer (ITS) region of rDNA and the β-tubulin gene were performed using ITS1 and ITS4 and Tub1 and Tub2 primer sets (4). Sequencing of the PCR products in both directions was conducted by IonTek Inc. (Istanbul, Turkey) in an ABI PRISM automated sequencer. The obtained sequences were compared with those in the GenBank and Phytophthora database using BLAST search. On the basis of morphological features and molecular analyses, the two isolates were identified as Phytophthora syringae. Morphological characteristics on carrot agar were identical with the description of P. syringae (2). At 20°C, colonies reached 7 cm in diameter after 1 week. Sporangia were semipapillate to non-papillate, ovoid, with average length of 59 μm (SD ± 2.8) (range 58 to 70 μm). Oogonia were 38 μm (SD ± 5.4) in diameter (range 30 to 47 μm) with paragynous antheridia. The morphological identification was confirmed by sequence comparison at GenBank with 99% homology for both ITS and β-tubulin. The ITS sequences of the two isolates were deposited in GenBank with the accession nos. KF430614 and KF944377. Under-bark inoculation tests with mycelia plugs were conducted with both isolates of P. syringae at 18°C in a growth chamber on a total of six 1-year-old shoots cut from two C. libani trees. Lesions with an average length of 19 mm (SD ± 6) developed after 10 days. P. syringae was consistently re-isolated from the margins of necrotic tissues. Control shoots remained symptomless. To our knowledge, this is the first report of damage caused by P. syringae on C. libani seedlings in forest nursery in Turkey. References: (1) T. Çalışkan. Pages 109-130 in: Proceedings of Workshop “Hızlı gelişen türlerle ilgili rapor,” Ankara, Turkey, 1998. (2) T. Jung et al. Eur. J. For. Pathol. 26:253, 1996. (3) T. Jung et al. Mycol. Res. 107:772, 2003. (4) L. P. N. M. Kroon et al. Fung. Genet. Biol. 41:766, 2004.

Download Full-text

First Report of Septoria Leaf Spot of Lavandin Caused by Septoria lavandulae in Croatia

Plant Disease ◽

10.1094/pdis-07-13-0735-pdn ◽

2014 ◽

Vol 98 (2) ◽

pp. 282-282

Author(s):

K. Vrandečić ◽

J. Ćosić ◽

D. Jurković ◽

I. Stanković ◽

A. Vučurović ◽

...

Keyword(s):

Leaf Spot ◽

Antibacterial Properties ◽

Morphological Characteristics ◽

Its Region ◽

Morphological Identification ◽

Conidial Suspension ◽

Necrotic Tissue ◽

First Report ◽

Initial Symptoms ◽

Septoria Leaf Spot

Lavandula × intermedia Emeric ex Loiseleur, commonly known as lavandin, is an aromatic and medicinal perennial shrub widely and traditionally grown in Croatia. The lavandin essential oil is primarily used in perfumery and cosmetic industries, but also possesses anti-inflammatory, sedative, and antibacterial properties. In June 2012, severe foliar and stem symptoms were observed on approximately 40% of plants growing in a commercial lavandin crop in the locality of Banovo Brdo, Republic of Croatia. Initial symptoms on lower leaves included numerous, small, oval to irregular, grayish brown lesions with a slightly darker brown margin of necrotic tissue. Further development of the disease resulted in yellowing and necrosis of the infected leaves followed by premature defoliation. Similar necrotic oval-shaped lesions were observed on stems as well. The lesions contained numerous, dark, sub-globose pycnidia that were immersed in the necrotic tissue or partly erumpent. Small pieces of infected internal tissues were superficially disinfected with 50% commercial bleach (4% NaOCl) and placed on potato dextrose agar (PDA). A total of 10 isolates from leaves and five from stems of lavandin formed a slow-growing, dark, circular colonies with raised center that produced pycnidia at 23°C, under 12 h of fluorescent light per day. All 15 recovered isolates formed uniform hyaline, elongate, straight or slightly curved conidia with 3 to 4 septa, with average dimensions of 17.5 to 35 × 1.5 to 2.5 μm. Based on the morphological characteristics, the pathogen was identified as Septoria lavandulae Desm., the causal agent of lavender leaf spot (1,2). Pathogenicity of one selected isolate (428-12) was tested by spraying 10 lavandin seedlings (8 weeks old) with a conidial suspension (106 conidia/ml) harvested from a 4-week-old monoconidial culture on PDA. Five lavandin seedlings, sprayed with sterile distilled water, were used as negative control. After 5 to 7 days, leaf spot symptoms identical to those observed on the source plants developed on all inoculated seedlings and the pathogen was successfully re-isolated. No symptoms were observed on any of the control plants. Morphological identification was confirmed by amplification and sequencing of the internal transcribed spacer (ITS) region of rDNA (3). Total DNA was extracted directly from fungal mycelium with a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and PCR amplification performed with primers ITS1F/ITS4. Sequence analysis of ITS region revealed at least 99% identity between the isolate 428-12 (GenBank Accession No. KF373078) and isolates of many Septoria species; however, no information was available for S. lavandulae. To our knowledge, this is the first report of Septoria leaf spot of lavandin caused by S. lavandulae in Croatia. Since the cultivation area of lavandin plants has been increasing in many continental parts of Croatia, especially in Slavonia and Baranja counties, the presence of a new and potentially harmful disease may represent a serious constraint for lavandin production and further monitoring is needed. References: (1) T. V. Andrianova and D. W. Minter. IMI Descriptions of Fungi and Bacteria, 142, Sheet 1416, 1999. (2) R. Bounaurio et al. Petria 6:183, 1996. (3) G. J. M. Verkley et al. Mycologia 96:558, 2004.

Download Full-text

First Report of Alternaria alternata Causing Leaf Spot on Bruguiera gymnorrhiza in China

Plant Disease ◽

10.1094/pdis-07-14-0755-pdn ◽

2015 ◽

Vol 99 (2) ◽

pp. 286-286 ◽

Cited By ~ 2

Author(s):

Q.-L. Lin ◽

H.-R. Su ◽

H. He

Keyword(s):

Leaf Spot ◽

Direct Sequencing ◽

Morphological Characteristics ◽

Its Region ◽

Average Diameter ◽

Morphological Observation ◽

Leaf Spot Disease ◽

Bruguiera Gymnorrhiza ◽

First Report ◽

Mangrove Tree

Bruguiera gymnorrhiza (L.) Savigny is an important mangrove tree species that grows in the intertidal regions of the tropical and subtropical coastlines. In a survey conducted in March 2014, a leaf spot disease on this plant was observed in Sea View Promenade in Zhanjiang, Guangdong Province, China. Symptoms on leaves initially appeared as small circular to irregular, dark brown, necrotic, sunken spots with an average diameter of 4 to 7 mm. The spots gradually enlarged in size, becoming irregular, or remained circular with concentric rings or zones. In the latter, the spots coalesced, and the leaves withered, dried, and fell from the plants. Leaf tissues (3 × 5 mm), cut from the margins of lesions, were surface-disinfected, placed on potato dextrose agar (PDA), and incubated at 28°C with a 12-h photoperiod. Five fungi (MLL1 to MLL5) with different morphological characteristics were obtained. To fulfill Koch's postulates, wounded and nonwounded leaves were inoculated. Fresh wounds were made with a sterile needle on 10 detached leaves and 10 leaves on five living plants for fungi MLL1 to MLL5 independently. Mycelial plugs of each fungus were applied to wounded and nonwounded leaves. For the control, 10 leaves on five living plants were inoculated with agar plugs in a similar manner, to both wounded and nonwounded leaves. All treatments were incubated in a humid chamber in the dark at 28°C. Leaf spots identical to those observed in the field were observed on the wounded leaves inoculated with fungus MLL3 after 3 to 4 days, while the other four fungi and the control remained symptomless. The 10 nonwounded leaves inoculated with fungus MLL3 were also infected after 5 days. The fungus, with the same colony and conidial morphology as MLL3, was re-isolated from the affected leaves. The pathogenic test was repeated three times under the same conditions. Hyphal tips of MLL3 were transferred to PDA for morphological observation. Colonies of white-to-dark-gray mycelia, black on the underside, formed on PDA. The colonies were further identified as Alternaria sp., based on the dark brown, obclavate to obpyriform catenulate conidia with longitudinal and transverse septa tapering to a prominent beak attached in chains on a simple and short conidiophore (3). Conidia varied from 22.5 × 40.26 to 3.95 × 5.79 μm and had three to eight transverse and zero to four longitudinal septa, with a beak length of 0 to 7.25 μm. For molecular identification, PCR was carried out using internal transcribed spacer (ITS) region primers ITS1/ITS4, partial sequences of the beta tubulin gene primers Bt1a-Bt1b (1), and A. alternata species-specific primers AAF2/AAR3 (2). The PCR products were subjected to direct sequencing. The resulting sequences were compared against the GenBank nucleotide database by using a BLAST alignment, which revealed that MLL3 had 99 to 100% identity with A. alternata for the ITS, Bt1a-Bt1b, and AAF2/AAR3 regions (GenBank Accession Nos. KF669893, GQ240308, and KJ716876, respectively). Sequences for MLL3 were deposited in GenBank under accession numbers KJ767515, KJ921779, and KJ921778. According to both morphological and sequence analyses, the pathogen of the leaf spot of B. gymnorrhiza was identified as A. alternata. To our knowledge, this is the first report of A. alternata on leaves of B. gymnorrhiza in China. This pathogen could cause serious foliar damage and threaten the survival, growth, and fitness of the local B. gymnorrhiza community. References: (1) N. L. Glass and G. C. Donaldson. Appl. Environ. Microbiol. 61:1323, 1995. (2) P. Konstantinova et al. Mycol. Res. 106:23, 2002. (3) E. G. Simmons. Alternaria: An identification Manual. CBS Fungal Biodiversity Center, Utrecht, Netherlands, 2007.

Download Full-text

First Report of Black Spot of Acanthus ilicifolius Caused by Fusarium solani in China

Plant Disease ◽

10.1094/pdis-04-14-0432-pdn ◽

2014 ◽

Vol 98 (10) ◽

pp. 1438-1438 ◽

Cited By ~ 1

Author(s):

H.-R. Su ◽

H. He ◽

Q.-Z. Huang ◽

N.-H. Lu ◽

Y.-B. Zhang

Keyword(s):

Fusarium Solani ◽

Morphological Characteristics ◽

Black Spot ◽

Morphological Observation ◽

Molecular Characteristics ◽

Beta Tubulin ◽

First Report ◽

Acanthus Ilicifolius ◽

Black Spots ◽

Initial Symptoms

Acanthus ilicifolius (family Acanthaceae) grows mainly in tropical coastal areas and is an important medicinal plant that can be used to treat asthma, rheumatism, etc. In July 2013, symptoms of black spots on the leaves of A. ilicifolius were observed in the Mangrove Conservation Area of Shenzhen Futian (22°32′ N, 114°03′ E) and Leizhou peninsula (20°12′~21°35′ N, 109°30′~110°55′ E), Guangdong Province, China. Initial symptoms of the disease were a small, dark brown spots (4 to 5 × 4 to 6 mm) surrounded by a yellow halo (1 to 2 mm in diameter), that would later extend to round or irregular black spots. Leaves eventually turned chlorotic and plants defoliated. Tissues from symptomatic leaves were excised, surface sterilized with 75% ethanol solution (v/v) for 20 s, soaked in 0.1% HgCl2 solution for 45 s, rinsed three times in sterile water, cut into small pieces (2 to 3 mm), plated on potato dextrose agar (PDA), and incubated 3 to 5 days at 28°C without light. Four isolates named from LSL-1 to LSL-4 with different morphological characteristics were obtained. To fulfill Koch's postulates, wounded and non-wounded leaves were inoculated. Fresh wounds were made with a sterile needle on detached leaves and on living plants. Mycelial plugs of each isolate were applied and covered with a piece of wet cotton to maintain moisture. For the control, the healthy leaves were inoculated with PDA plugs. All treatments were incubated at room temperature. Black spots were observed on the wounded leaves inoculated with isolate LSL-1 after 3 days, while the other three isolates and the control remained symptomless, and the pathogen similar to LSL-1 was re-isolated from the diseased leaves. Non-wounded leaves didn't become infected. The pathogenic test was repeated three times with the same conditions, and it was confirmed that LSL-1 was the pathogen causing the black spot of A. ilicifolius. Identification of the pathogen was conducted using morphological and molecular characteristics. Hyphal tips of LSL-1 were transferred to PDA medium in petri dishes for morphological observation. Two types of conidia were observed. The macroconidia were cylindrical to slightly curved, falciform shaped, with two to four septa, and measured 39 to 45 × 4.7 to 5.0 μm. The microconidia were oval to kidney shaped, single celled, 8 to 10 × 2.5 to 3.5 μm. Chlamydospores were also observed, produced singly or in pairs. Based on morphology (1,4), the isolate was tentatively identified as Fusarium solani. For molecular identification, the internal transcribed spacer (ITS) of ribosomal DNA, beta-tubulin gene, and translation elongation factor 1-alpha (EF-1α) gene was amplified using the ITS1/ITS4 (5), ITS4/ITS5 (5), T1/T2 (2) and EF1/EF2 (3) primer pairs. The gene sequences were deposited in GenBank (KJ720639 for the ITS1/ITS4 region, KF826493 for the ITS4/ITS5 region, KJ720638 for the beta-tubulin, and KF826492 for EF-1α region) and showed 99% identity to the F. solani strains (AY633746 for ITS1/ITS4 region, AM412637 for ITS4/ITS5 region, KF255996 for beta-tubulin region, DQ246859 for EF-1α region). According to these results, the pathogen of black spot of A. ilicifolius was identified as F. solani. To the best of our knowledge, this is the first report of F. solani causing black spot of A. ilicifolius in China. References: (1) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell, Ames, IA, 2006. (2) K. O'Donnell and E. Cigelnik. Mol. Phylogenet. Evol. 7:103, 1997. (3) K. O'Donnell et al. Proc. Natl. Acad. Sci. USA. 95:2044, 1998. (4) B. A. Pérez et al. Plant Dis. 91:1053, 2007. (5) A. W. Zhang et al. Plant Dis. 81:1143, 1997.

Download Full-text

First Report of Verticillium Wilt Caused by Verticillium dahliae on Figmarigold in Italy

Plant Disease ◽

10.1094/pdis-94-1-0129b ◽

2010 ◽

Vol 94 (1) ◽

pp. 129-129

Author(s):

A. Garibaldi ◽

D. Bertetti ◽

P. Pensa ◽

M. L. Gullino

Keyword(s):

Verticillium Wilt ◽

Cross Sections ◽

Vascular Tissue ◽

Morphological Characteristics ◽

Ground Cover ◽

Its Region ◽

Peat Moss ◽

Conidial Suspension ◽

First Report ◽

Initial Symptoms

Lampranthus spp. N.B. Brown (figmarigold) belongs to the Aizoaceae family and is grown as a ground cover in gardens or as a potted plant. In January 2009, severe outbreaks of a previously unknown wilt were observed at a commercial farm in Liguria (northern Italy) where 7-month-old potted plants were grown outdoors in a mix of peat/clay/pumice at pH 6.5. In cultivars with pink flowers, 12% of plants were affected, while only a few cultivars with red flowers were diseased. Initial symptoms were yellowing of external leaves and brown or black streaks in the vascular tissue of roots, crown, and leaves. Subsequently, infected tissues wilted and stopped growing, stems and leaves appeared desiccated, and infected plants died. Stems of 10 pink-flowered plants were severed with a knife, cut ends sealed with wax, and surfaces disinfected with 1% sodium hypochlorite. Cross-sections (1 mm long) through symptomatic vascular tissue were plated onto potato dextrose agar. After 10 days at 22°C, 90% of the stems tested positive for Verticillium. Irregular, dark microsclerotia, 22 to 128 × 13 to 66 (average 51 × 29) μm, developed in hyaline hyphae after 10 days of growth at 22 ± 1°C (12-h photoperiod). Hyaline, elliptical, single-celled conidia, 2.9 to 4.8 × 1.3 to 2.4 (average 4.1 × 1.7) μm, developed on verticillate conidiophores. On the basis of these morphological characteristics, the fungus was identified as V. dahliae (3). The internal transcribed spacer (ITS) region of rDNA was amplified using primers ITS4/ITS6 (2) and sequenced. BLASTn analysis (1) of the 476-bp segment showed a 100% homology with the sequence of V. dahliae. The nucleotide sequence has been assigned GenBank Accession No. GQ 149479. Pathogenicity tests were performed twice using five 40-day-old plants of a pink-flower cultivar of a Lampranthus sp. grown in 1-liter pots containing a 50:20:20:10 mix of peat moss/pumice/pine bark cortex/clay. The substrate was infested with a conidial suspension (1.0 × 107/ml) of one isolate of V. dahliae recovered from infected plants. Inoculum (50 ml) were added to each pot, drenching the top of the soil. Noninoculated plants served as controls. Plants (five per treatment) were maintained in a glasshouse at daily average temperatures between 20 and 26°C and at 50 to 70% relative humidity. The first wilt symptoms and a vascular discoloration in the roots, crown, and veins developed 30 days after inoculation. V. dahliae was consistently reisolated. Noninoculated plants remained healthy. In a second test, the susceptibility of purple-, white-, yellow-, red-, and orange-flowered cultivars was tested. Ten rooted cuttings of each cultivar were inoculated as described above. The severity of Verticillium wilt was evaluated and each cultivar was classified as resistant, partially resistant, average susceptible, susceptible, or highly susceptible. All tested cultivars were susceptible or highly susceptible to Verticillium. Only the purple cultivar showed an average susceptibility. To our knowledge, this is the first report of Verticillium wilt on Lampranthus spp. in Italy as well as worldwide. Today, the economic importance of Verticillium wilt on figmarigold in Italy is still limited. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) M. A. Innis et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990. (3) G. F. Pegg and B. L. Brady. Verticillium Wilts. CABI Publishing, Wallingford, UK, 2002.

Download Full-text

First Report of Phoma rhei as a Pathogen of Rheum rhabarbarum in China

Plant Disease ◽

10.1094/pdis-01-14-0019-pdn ◽

2014 ◽

Vol 98 (8) ◽

pp. 1154-1154 ◽

Cited By ~ 1

Author(s):

Y. N. Liu ◽

N. Nan ◽

B. H. Lu ◽

W. Y. Xia ◽

X. Y. Wu ◽

...

Keyword(s):

Early Stage ◽

Morphological Characteristics ◽

Its Region ◽

Control Treatment ◽

Perennial Herb ◽

Morphological Identification ◽

First Report ◽

Plastic Bags ◽

Initial Symptoms ◽

Necrotic Spots

Rheum rhabarbarum L., rhubarb, is a perennial herb planted mainly in Hebei, Hubei, Shanxi, Heilongjiang, and Jilin provinces as well as Inner Mongolia, China. The plant grows about 1,000 meters above sea level (4), and is used widely in China to treat constipation and gout. From June to September 2012, a leaf spot was observed on R. rhabarbarum in the medicinal garden of Jilin Agricultural University, Changchun, Jilin Province, causing significant effects on the leaves of all infected plants. In the early stage of disease development, small red lesions were visible on infected leaves, which subsequently developed into irregularly shaped or circular necrotic spots, each with a light colored center, pink-red alternating concentric rings, and surrounded by a chlorotic halo. Some lesions became perforated in the center. Lesions ranged from 1 to 15 mm in diameter. Extensive spotting resulted in general browning and yellowing of entire leaves. As lesions enlarged and coalesced, some leaves died from the margin inwards. Lesions on the stem were fusiform and sunken. Small pieces of diseased leaves and stems were surface-disinfested in 75% ethanol for 60 s, rinsed twice in distilled water, dried, and plated on potato dextrose agar (PDA). A Phoma species was isolated that produced a gray or dark gray colony after 5 to 7 days. The isolate was transferred to oatmeal agar (OA) (3). Pycnidia were dark brown to black, globose to subglobose, and 121 to 354 × 100 to 262 μm. Conidia were ellipsoidal or reniform, colorless, unicellular, and 3.8 to 6.5 × 1.7 to 4.1 μm. On the basis of these characteristics, the fungus was identified as Phoma rhei (1). A PCR assay with the ITS4 and ITS5 primers was used to amplify the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) (2). The amplified product (567 bp) was sequenced and the sequence submitted to GenBank (Accession No. KF531831). The ITS sequence exhibited 99% identity to that of a P. rhei isolate in GenBank (GU237743.1), confirming the morphological identification. Pathogenicity of eight isolates on rhubarb was confirmed by spraying a spore suspension (1 × 106 spores/ml) produced on PDA on the leaves of each 6-year-old R. rhabarbarum (cv. Boyedahuang) plant. Each isolate was inoculated onto five plants, and five plants were sprayed similarly with sterilized water as a control treatment. The plants were then covered with plastic bags for 48 h, and kept in a greenhouse (20 to 30°C with a 12-h photoperiod/day). Initial symptoms on inoculated leaves were observed 3 to 4 days after inoculation, while the control plants remained healthy. Re-isolations from lesions on the inoculated leaves, using the same protocol as the original isolations, produced fungal colonies with the same morphological characteristics as the original isolates of P. rhei, but no fungi were re-isolated from the control plants. This fungus has been found on R. rhaponticum in New Zealand (1), but to our knowledge this is the first report of P. rhei on R. rhabarbarum in China. References: (1) G. H. Boerema et al. Phoma Identification Manual. Diffferentiation of Specific and Infra-Specific Taxa in Culture. CABI Publishing. Wallingford, UK, 2004. (2) D. E. L. Cooke et al. Mycol. Res. 101:667, 1997. (3) Z. D. Fang. Research Method of Phytopathology. China Agricultural Press (In Chinese), 1998. (4) A. J. Li et al. Flora Reipublicae Popularis Sinicae. Tomus 25:171, 1998.

Download Full-text

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.

Download Full-text

First Report of Verticillium Wilt Caused by Verticillium dahliae on Rudbeckia fulgida (Orange Coneflower) in Italy

Plant Disease ◽

10.1094/pdis-92-9-1367c ◽

2008 ◽

Vol 92 (9) ◽

pp. 1367-1367 ◽

Cited By ~ 1

Author(s):

A. Garibaldi ◽

D. Bertetti ◽

M. L. Gullino

Keyword(s):

United States ◽

Verticillium Wilt ◽

Verticillium Dahliae ◽

Vascular Tissue ◽

Morphological Characteristics ◽

Its Region ◽

The United States ◽

Conidial Suspension ◽

First Report ◽

Initial Symptoms

Rudbeckia fulgida (common name orange coneflower) is an herbaceous perennial (Asteraceae) grown in full sun in perennial borders in gardens. At the end of the summer of 2007, in a public garden located in Turin (northern Italy), symptoms of vascular wilt and stunting were observed on approximately 80% of the plants grown in a mixed border. Initial symptoms were yellowing of external leaves and brown or black streaks in the vascular tissue of roots, crown, and leaves. A fungus was consistently and readily isolated on potato dextrose agar from symptomatic vascular tissue previously disinfested in 1% sodium hypochloride. Ovoid, dark microsclerotia, 41 to 108 μm, developed in hyaline hyphae after 10 days of growth at 23°C (12 h of light and 12 h of dark). Hyaline, elliptical, single-celled conidia, 3.2 to 7.3 × 2.1 to 3.7 (average 4.7 × 2.8) μm, developed on verticillate conidiophores. On the basis of these morphological characteristics, the fungus was identified as Verticillium dahliae (4). The internal transcribed spacer (ITS) region of rDNA was amplified using primers ITS4/ITS6 and sequenced. BLASTn analysis (1) of the 530 bp showed a 100% homology with the sequence of V. dahliae. The nucleotide sequence has been assigned GenBank Accession No. EU 627007. Healthy, 30-day-old R. fulgida plants were grown in a steam-disinfested mix of sphagnum peat:pomix:pine bark:clay (50:20:20:10) infested with a conidial suspension (1.5 × 106/ml) of three isolates of V. dahliae isolated from infected plants. Noninoculated plants served as controls. Plants (16 per treatment) were grown in pots (3 liter vol) and maintained in a glasshouse at temperatures between 22 and 25°C and relative humidity between 50 and 70%. First wilt symptoms and vascular discoloration in the roots, crown, and veins developed 17 days after inoculation. Noninoculated plants remained healthy. The pathogenicity tests were carried out twice. To our knowledge, this is the first report in Italy of Verticillium wilt on R. fulgida. Wilts caused by V. dahliae on R. laciniata in Poland (3) and V. albo-atrum on R. hirta in the United States (2) were previously reported. The importance and economic impact of this disease is currently limited but may increase because of the popularity of Rudbeckia spp. in private and public parks. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) D. F. Farr et al. Fungi on Plants and Their Products in the United States. The American Phytopathological Society, St Paul, MN, 1989. (3) B. Leski. Rocz. Nauk Roln. 253, 1974. (4) G. F. Pegg and B. L. Brady. Verticillium Wilts. CABI Publishing, Wallingford, UK, 2002.

Download Full-text