First report that Colletotrichum aenigma causes leaf spots on Aquilaria sinensis in China

Aquilaria sinensis (Lour.) Spreng, also known as eaglewood, belongs to the Thymelaeaceae family and has a considerably high medicinal value. It has been enlisted as the class II national key protective plant. In June 2019, about 15 percent of A. sinensis treelets in a forest area of China's Hainan province were observed to have the anthracnose symptoms. The diseased spots on leaves of A. sinensis treelets were usually round or irregular with pale yellow edges. The color of the center of the lesion was firstly light brown and then black or yellowish-brown. Small pieces of tissue from the edge of the leaf spots were surface sterilized in 75% alcohol for the 60s, washed twice with sterile distilled water, and then cultivated at 28 °C in darkness on potato dextrose agar (PDA) medium. One fungus was systematically isolated to get pure cultures. The culturing of the three isolates was carried out in PDA media at 28 °C for a week. The average diameter of the collateral colony was 6.80 ±0.60 cm. Initially, the fungal colonies were white aerial mycelium and the central area of the colonies slowly turned jacinth. After seven days, the central mycelium turns grayish-green and the colonies’ undersurfaces were grey to white. The colony's surfaces were fluffy and round with smooth edges. Conidia were cylindrical, smooth, and transparent, with a slight indentation in the middle and uneven distribution of small particles inside, 12.5–20.6×3.5–6.8 µm (ave=15.9±1.40×5.18±1.07, n=50). Appressoria were typically elliptic or irregular and brown to dark brown. The isolates were characterized as Colletotrichum gloeosporioides species complex on the basis of the conidial morphology and culture representation, (Deng et al. 2017; Weir et al. 2012). To further verify the identification of the species, CX-0301, the isolated representative strains were extracted for genomic DNA. mating type 1-2-1 (Mat-1-2-1) ApMat, actin (ACT) gene, chitin synthase (CHS), and beta-tubulin (TUB2) gene were amplified using the primer pairs VcaMat-5F/VcaMat-5R, ACT-512F/ACT-783R, CHS-1-79F/CHS-1-354R, and TUB2-T1/Bt2b, respectively (Damm et al. 2012; Du et al. 2005). The homologous sequences of MN310694, MN310693, MN310692, and MN310691 were submitted to GenBank. These genes have ≥a 97% sequence similarity to the genes of Colletotrichum aenigma (MG717319.1, MG717317.1, MH476565.1, MH853679.1, respectively) in GenBank. These morphological and molecular characteristics identified that the pathogen is C. aenigma. (Weir et al. 2012). To further verify the isolated pathogen, the pathogenicity test was performed on uninfected healthy 2-year-old eaglewood seedlings. The conidial suspension (1×106 conidia/ml) of 5ml was sprayed on both surfaces of 10 leaves of plants of the same age and height and the controls were treated solely with distilled water (Deng et al. 2017). Upon completion of inoculation, plants were kept under greenhouse conditions with an assigned temperature of 28 ± 2°C while keeping relative humidity to 90% on a 12-h fluorescent light/dark regime. Anthracnose-like symptoms were observed 6 days postinoculation. The control plant tissues remained healthy. Follow up reisolation of C. enigma culture was obtained in PDA agar plates from leaf infected lesions, and the morphological features were found to be consistent with that of CX-0301 isolate, satisfying Koch's postulates. Based on the characterized information, it is the first report of Colletotrichum aenigma responsible for causing leaf spots on Aquilaria sinensis in China. Thereby, this provides a theoretical reference for the research and control of anthracnose on A. sinensis.

Download Full-text

First Report of Brown Blight Disease Caused by Colletotrichum gloeosporioides on Camellia sinensis in Anhui Province, China

Plant Disease ◽

10.1094/pdis-08-13-0896-pdn ◽

2014 ◽

Vol 98 (2) ◽

pp. 284-284 ◽

Cited By ~ 16

Author(s):

M. Guo ◽

Y. M. Pan ◽

Y. L. Dai ◽

Z. M. Gao

Keyword(s):

Camellia Sinensis ◽

Colletotrichum Gloeosporioides ◽

Tea Plant ◽

Anhui Province ◽

Molecular Characteristics ◽

Fluorescent Light ◽

Distilled Water ◽

Conidial Suspension ◽

First Report ◽

Twig Blight

Yellow Mountain fuzz tip, a cultivar of Camellia sinensis (L.) Kuntze, is commonly grown in the Yellow Mountain region in Anhui Province of China. During 2011 to 2012, leaf and twig blight on tea plants occurred from July to September in growing regions. Symptoms of blight on leaves of infected plants were detected in 30 to 60% of the fields visited and up to 500 ha were affected each year. Symptoms began as small, water-soaked lesions on young leaves and twigs and later became larger, dark brown, necrotic lesions, 1 to 3 mm in diameter on leaves and 2 to 5 mm long on twigs. To determine the causal agent, symptomatic leaf tissue was collected from plants in Gantang and Tangkou townships in September 2012. Small pieces of diseased tea leaves and twigs were surface-disinfested in 2% NaClO for 3 min, rinsed twice in distilled water, plated on potato dextrose agar, and incubated at 28°C for 5 days. Eleven isolates were recovered and all cultures produced white-to-gray fluffy aerial hyphae and were dark on the reverse of the plate. The hyphae were hyaline, branching, and septate. Setae were 2- to 3-septate, dark brown, acicular, and 78.0 to 115.0 μm. Conidiogenous cells were hyaline, short, branchless, cylindrical, and 11.3 to 21.5 × 4.2 to 5.3 μm. Conidia were hyaline, aseptate, guttulate, cylindrical, and 12.5 to 17.3 × 3.9 to 5.8 μm. Appresoria were ovate to obovate, dark brown, and 8.4 to 15.2 × 7.8 to 12.9 μm. DNA was amplified using the rDNA-ITS primer pair ITS4/ITS5 (3), glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) primer pair GDF/GDR (2) and beta-tubulin 2 gene (Tub2) primer pair Btub2Fd/Btub4Rd (4). Sequences (GenBank Accession Nos. KC913203, KC913204, and KC913205) of the 11 isolates were identical and revealed 100% similarity to the ITS sequence of strain P042 of Colletotrichum gloeosporioides (EF423527), 100% identity to the GAPDH of isolate C07009 of C. gloeosporioides (GU935860), and 99% similarity to Tub2 of isolate 85 of C. gloeosporioides (AJ409292), respectively. Based on the above data, the 11 isolates were identified as C. gloeosporioides (Penz.) Penz. & Sacc. To confirm pathogenicity, Koch's postulate was performed and 4 ml of conidial suspension (1 × 105 conidia/ml) of each of the 11 isolates was sprayed on five leaves and five twigs per plant on four 12-month-old Yellow Mountain fuzz tip plants. Control plants were sprayed with distilled water. The inoculated plants were maintained at 28°C in a greenhouse with constant relative humidity of 90% and a 12-h photoperiod of fluorescent light. Brown necrotic lesions appeared on leaves and twigs after 7 days, while the control plants remained healthy. The experiments were conducted three times and the fungus was recovered and identified as C. gloeosporioides by both morphology and molecular characteristics. Tea plant blight caused by C. gloeosporioides was identified in Brazil (1), but to our knowledge, this is the first report of C. gloeosporioides causing tea leaf and twig blight on Yellow Mountain fuzz tip plants in Anhui Province of China. References: (1) M. A. S. Mendes et al. Page 555 in: Embrapa-SPI/Embrapa-Cenargen, Brasilia, 1998. (2) M. D. Templeton et al. Gene 122:225, 1992. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990. (4) J. H. C. Woudenberg et al. Persoonia 22:56, 2009.

Download Full-text

First Report of Leaf Spot on Industrial Hemp (Cannabis sativa L.) Caused by Alternaria alternata in China

Plant Disease ◽

10.1094/pdis-01-21-0130-pdn ◽

2021 ◽

Author(s):

Lili Tang ◽

Xixia Song ◽

Liguo Zhang ◽

Jing Wang ◽

Shuquan Zhang

Keyword(s):

Leaf Spot ◽

Cannabis Sativa ◽

Leaf Spot Disease ◽

Molecular Characteristics ◽

Pathogenicity Test ◽

Conidial Suspension ◽

Sterile Water ◽

First Report ◽

Leaf Spots ◽

Industrial Hemp

Industrial hemp is an economically important plant with traditional uses for textiles, paper, building materials, food and medicine (Li 1974; Russo et al. 2008; Zlas et al. 1993). In August 2020, an estimated 80% of the industrial hemp plants with leaf spots were observed in greenhouse in Minzhu town, Harbin City, Heilongjiang Province, China (45.8554°N, 126.8167°E), resulting in yield losses of 20%. Leaf symptoms began as small spots on the upper surface of leaves and gradually developed into brown spots with light yellow halos. These irregular spots expanded gradually and eventually covered the entire leaf; the center of the spots was easily perforated. To identify the pathogen, 20 diseased leaves were collected, and small sections of (3 to 5 mm) were taken from the margins of lesions of infected leaves. The pieces were sterilized with 75% alcohol for 30 s, a 0.1% mercuric chloride solution for 1 min, and then rinsed three times with sterile water. Samples were then cultured on potato dextrose agar at 28℃ in darkness for 4 days. A single-spore culture was obtained by monosporic isolation. Conidiophores were simple or branched, straight or flexuous, brown, and measured 22 to 61 μm long × 4 to 5 μm wide (n = 50). Conidia were solitary or in chains, brown or dark brown, obclavate, obpyriform or ellipsoid. Conidia ranged from 23 to 55 μm long × 10 to 15 μm wide (n = 50) with one to eight transverse and several longitudinal septa. For molecular identification (Jayawardena et al. 2019), genomic DNA of pathogenic isolate (MZ1287) was extracted by a cetyltrimethylammonium bromide protocol. Four gene regions including the rDNA internal transcribed spacer (ITS), glyceraldehyde-3-phosplate dehydrogenase (GAPDH), translation elongation factor 1-alpha (TEF1) and RNA polymerase II beta subunit (RPB2) were amplified with primers ITS1/ITS4, GDF1/GDR1, EF1-728F/EF1-986R and RPB2-5F/RPB2-7cR, respectively (White et al. 1990). Resulting sequences were deposited in GenBank with accession numbers of MW272539.1, MW303956.1, MW415414.1 and MW415413.1, respectively. A BLASTn analysis showed 100% homology with A. alternata (GenBank accession nos. MN615420.1, MH926018.1, MN615423.1 and KP124770.1), respectively. A neighbor-joining phylogenetic tree was constructed by combining all sequenced loci in MEGA7. The isolate MZ1287 clustered in the A. alternata clade with 100% bootstrap support. Thus, based on morphological (Simmons 2007) and molecular characteristics, the pathogen was identified as A. alternata. To test pathogenicity, leaves of ten healthy, 2-month-old potted industrial hemp plants were sprayed using a conidial suspension (1×106 spores/ml). Control plants were sprayed with sterile water. All plants were incubated in a greenhouse at 25℃ for a 16 h light and 8 h dark period at 90% relative humidity. The experiment was repeated three times. After two weeks, leaf spots of industrial hemp developed on the inoculated leaves while the control plants remained asymptomatic. The A. alternata pathogen was re-isolated from the diseased leaves on inoculated plants, fulfilling Koch's postulates. Based on morphology, sequencing, and pathogenicity test, the pathogen was identified as A. alternata. To our knowledge, this is the first report of A. alternata causing leaf spot disease of industrial hemp (Cannabis sativa L.) in China and is worthy of our attention for the harm it may cause to industrial hemp production.

Download Full-text

First Report of Alternaria Blight of Potato Caused by Alternaria tenuissima in China

Plant Disease ◽

10.1094/pdis-08-12-0763-pdn ◽

2013 ◽

Vol 97 (9) ◽

pp. 1246-1246 ◽

Cited By ~ 7

Author(s):

H. H. Zheng ◽

X. H. Wu

Keyword(s):

Solanum Tuberosum L ◽

Histone Gene ◽

Gansu Province ◽

Molecular Characteristics ◽

Fluorescent Light ◽

Distilled Water ◽

Conidial Suspension ◽

Species Identity ◽

First Report ◽

Alternaria Blight

Potato (Solanum tuberosum L.) is grown worldwide as a major food crop. Potato early blight is an important disease caused by Alternaria solani (4). In 2011, diseased potato leaves with blight symptoms were collected from 21 sites (incidence averaged 60% for about 2,000 ha of potato fields examined) in Gansu Province, northwest China. Small pieces of tissue taken from the margin between healthy and diseased tissues were surface-disinfected in 0.3% NaOCl for 2 min, rinsed with sterilized, distilled water, then placed on potato dextrose agar (PDA) at 25°C in the dark. Two of 24 Alternaria isolates from single-spore cultures were identified preliminarily as A. tenuissima, and the remaining isolates as A. solani or A. alternata, based on morphological traits. Colony appearance on potato carrot agar (PCA) was loosely cottony under a day/night cycle of 8 h fluorescent light/16 h dark at 22°C for 7 days (3). The isolates were characterized by formation of unbranched conidial chains up to 12 conidia in length, with one or two lateral branches forming occasionally. Conidia were typically ovoid to obclavate, and ranged from 20.4 to 42.4 × 7.7 to 13.2 μm. Transverse septa and longitudinal septa of conidia varied from 1 to 6 and 0 to 2, respectively. Short conidiophores arose singly and were 15.1 to 76.8 μm long by 2.4 to 6.2 μm wide. The internal transcribed spacer (ITS) region of rDNA and partial coding sequence of a histone gene were amplified from genomic DNA of the two A. tenuissima isolates using the ITS1/ITS4 and H3-1a/H3-1b primers (2), respectively. The ITS sequences of the two isolates (GenBank Accession Nos. JX495165 and JX495166) were 100% identical to those of A. tenuissima strains sdau 07-100 and BL08-3 (GQ871507 and AB470887), as well as to other Alternaria species, but the partial histone gene sequences (JX495167 and JX495168) were 99% identical to those of A. tenuissima isolates CR27, MA1, MA6, and CN-L-01 (AF404622, AF404633, AF404634, and EF371552, respectively) with less similarity to those of other Alternaria spp. Therefore, the isolates were identified as A. tenuissima based on morphological and molecular characteristics. Pathogenicity tests were conducted by inoculating detached leaves (30 per isolate) from 45-day-old plants of potato cv. Favorita with 20 μl drops (one drop per leaf) of a conidial suspension containing 106 conidia/ml in sterilized, distilled water. Thirty control leaves were inoculated similarly with sterilized, distilled water. Inoculated leaves were incubated in chambers at 25°C and 90% RH with a 12-h photoperiod/day. After 7 days, symptoms on the inoculated leaves were similar to those naturally occurring on the original plants, and the two cultures were reisolated consistently from those leaves, and the species identity was confirmed by morphological and molecular characteristics, fulfilling Koch's postulates. The control leaves remained asymptomatic and Alternaria was not isolated from those leaves. Alternaria blight of potato caused by A. tenuissima was previously detected in Iran (1). To our knowledge, this is the first report of A. tenuissima causing blight on potatoes in China. References: (1) S. T. Ardestani et al. Iran. J. Plant Pathol. 45:83, 2010. (2) N. L. Glass and G. C. Donaldson. Appl. Environ. Microbiol. 61:1323, 1995. (3) E. G. Simmons. Alternaria. An Identification Manual. CBS Fungal Biodiversity Centre, Utrecht, the Netherlands, 2007. (4) J. E. van der Waals et al. Plant Dis. 88:959, 2004.

Download Full-text

First Report of Colletotrichum siamense causing Anthracnose on White Frangipani (Plumeria alba L.) in Malaysia

Plant Disease ◽

10.1094/pdis-12-20-2614-pdn ◽

2021 ◽

Author(s):

Siti Izera Ismail ◽

Nur Liyana Mohmad Zaiwawi ◽

Sumaiyah Abdullah ◽

Syari Jamian ◽

Norsazilawati Saad

Keyword(s):

Disease Incidence ◽

Morphological Characteristics ◽

Flowering Plant ◽

Molecular Characteristics ◽

Fluorescent Light ◽

Distilled Water ◽

Conidial Suspension ◽

Accurate Identification ◽

First Report ◽

Colletotrichum Siamense

Plumeria alba L. is a flowering plant in the family Apocynaceae and widely cultivated in Malaysia as a cosmopolitan ornamental plant. In January 2020, anthracnose lesions were observed on leaves of Plumeria alba planted in Agricultural Farm, Universiti Putra Malaysia, in Selangor state, Malaysia. The disease mainly affected the leaves with symptoms occurring with approximately a 60% disease incidence. Ten symptomatic leaves were sampled from 3 different trees in the farm. Symptoms initiated as small circular necrotic spots that rapidly enlarged into black lesions with pale brown borders. Diseased tissues (5×5 mm) were surface-sterilized with 70% ethanol for 1 min, rinsed three times with sterile distilled water, dried on sterile filter papers, plated on PDA and, incubated at 25 °C with a 12-h photoperiod. A total of seven single-spore isolates with similar colony morphologies were obtained from tissue samples. After 7 days, the colonies raised the entire margin and showed white-to-gray aerial mycelium, orange conidial masses in the center and appeared dark brown at the center of the reverse view. The conidia were 1-celled, hyaline, smooth-walled, cylindrical with narrowing at the center, averaged (13-15 μm × 3 - 4 μm) (n=40) in size. Morphological characteristics of the isolates were similar to those detailed in taxonomic description of Colletotrichum sp. (Prihastuti et al. 2009). For molecular identification, genomic DNA of two representative isolates, PL3 and PL4 was extracted from fresh mycelium using DNeasy Plant Mini Kit (Qiagen, USA). The internal transcribed spacer (ITS) region, actin (ACT) and calmodulin (CAL) genes were amplified using ITS5/ITS4 (White et al. 1990), ACT-512F/783R (Carbone and Kohn 1999) and CL1C/CL2C primer sets (Weir et al. 2012). A BLAST nucleotide search of GenBank using ITS sequences showed 100% identity to Colletotrichum siamense ex-type culture ICMP 18578 (GenBank accession no. JX010171). ACT and CAL sequences showed 100% identity with C. siamense ex-type isolate BPD-I2 (GenBank accession no. FJ907423 and FJ917505). The sequences were deposited in GenBank (ITS: accession nos. MW335128, MT912574), ACT: accession nos. MW341257, MW341256, CAL: accession nos. MW341255 and MT919260). Based on these morphological and molecular characteristics, the fungus was identified as C. siamense. Pathogenicity of PL3 and PL4 isolates was verified using four healthy detached leaves of Plumeria alba. The leaves were surface-sterilized using 70% ethanol and rinsed twice with sterile water before inoculation. The leaves (three inoculation sites/leaf) were wounded by puncturing with a sterile needle through the leaf cuticle and inoculated in the wound site with 10-μl of conidial suspension (1×106 conidia/ml) from 7-days-old culture on PDA. Four leaves were used as a control and were inoculated only with 10-μl of sterile distilled water. Inoculated leaves were kept in humid chambers for 2 weeks at 25 °C with 98% relative humidity on a 12-h fluorescent light/dark period. The experiment was repeated three times. Anthracnose symptoms were observed on all inoculated leaves after 3 days, whereas controls showed no symptoms. Fungal isolates from the diseased leaves showed the same morphological characteristics as isolates PL3 and PL4, confirming Koch’s postulates. C. siamense has been reported causing anthracnose on rose (Rosa chinensis) in China (Feng et al. 2019), Coffea arabica in Thailand (Prihastuti et al. 2009) and mango leaf anthracnose in Vietnam (Li et al. 2020). To our knowledge, this is the first report of Colletrotrichum siamense causing leaf anthracnose on Plumeria alba in Malaysia. Accurate identification of this pathogen provides a foundation in controlling anthracnose disease on Plumeria alba.

Download Full-text

First Report of Fusarium solani Causing Root Rot on Coptis chinensis in Southwestern China

Plant Disease ◽

10.1094/pdis-02-14-0164-pdn ◽

2014 ◽

Vol 98 (9) ◽

pp. 1273-1273 ◽

Cited By ~ 2

Author(s):

X.-M. Luo ◽

J.-L. Li ◽

J.-Y. Dong ◽

A.-P. Sui ◽

M.-L. Sheng ◽

...

Keyword(s):

Fusarium Solani ◽

Root Rot ◽

Southwestern China ◽

Molecular Characteristics ◽

Distilled Water ◽

Conidial Suspension ◽

Storage Roots ◽

Coptis Chinensis ◽

Causal Organism ◽

First Report

China is the world's largest producer country of coptis (Coptis chinensis), the rhizomes of which are used in traditional Chinese medicine. Since 2008, however, root rot symptoms, including severe necrosis and wilting, have been observed on coptis plants in Chongqing, southwestern China. Of the plants examined from March 2011 to May 2013 in 27 fields, 15 to 30% were covered with black necrotic lesions. The leaves of infected plants showed wilt, necrotic lesions, drying, and death. The fibrous roots, storage roots, and rhizomes exhibited brown discoloration and progressive necrosis that caused mortality of the infected plants. Infected plants were analyzed to identify the causal organism. Discoloration of the internal vascular and cortical tissues of the rhizomes and taproots was also evident. Symptomatic taproots of the diseased coptis were surface sterilized in 1% sodium hypochlorite for 2 min, rinsed in sterile distilled water for 2 min, and then air-dried in sterilized atmosphere/laminar flow. Small pieces of disinfested tissue (0.3 cm in length) were transferred to petri dishes containing potato dextrose agar (PDA) supplemented with 125 μg ml–1 streptomycin sulfate and 100 μg ml–1 ampicillin, and incubated for 5 days at 25°C with a 12-h photoperiod. Four distinct species of fungal isolates (HL1 to 4) derived from single spores were isolated from 30 plants with root rot symptoms collected from the study sites. To verify the pathogenicity of individual isolates, healthy coptis plants were inoculated by dipping roots into a conidial suspension (106 conidia/ml) for 30 min (15 plants per isolate), as described previously (1). Inoculated plants were potted in a mixture of sterilized quartz sand-vermiculite-perlite (4:2:1, v/v) and incubated at 25/18°C and 85 to 90% relative humidity (day/night) in a growth chamber with a daily 16-h photoperiod of fluorescent light. Plants dipped in sterile distilled water were used as controls. After 15 days, symptoms similar to those observed in the field were observed on all plants (n = 15) that were inoculated with HL1, but symptoms were not observed on plants inoculated with HL2, HL3, and HL4, nor on control plants. HL1 was re-isolated from symptomatic plants but not from any other plants. Morphological characterization of HL1 was performed by microscopic examination. The septate hyphae, blunt microconidia (2 to 3 septa) in the foot cell and slightly curved microconidia in the apical cell, and chlamydospores were consistent with descriptions of Fusarium solani (2). The pathogen was confirmed to be F. solani by amplification and sequencing of the ribosomal DNA internal transcribed spacer (rDNA-ITS) using the universal primer pair ITS4 and ITS5. Sequencing of the PCR product revealed a 99 to 100% similarity with the ITS sequences of F. solani in GenBank (JQ724444.1 and EU273504.1). Phylogenetic analysis (MEGA 5.1) using the neighbor-joining algorithm placed the HL1 isolate in a well-supported cluster (97% bootstrap value based on 1,000 replicates) with JQ724444.1 and EU273504.1. The pathogen was thus identified as F. solani based on its morphological and molecular characteristics. To our knowledge, this is the first report of root rot of coptis caused by F. solani in the world. References: (1) K. Dobinson et al. Can. J. Plant Pathol. 18:55, 1996. (2) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Oxford, 2006.

Download Full-text

First Report of Curvularia aeria on Etlingera linguiformis from Nagaland, India

Plant Disease ◽

10.1094/pdis-01-14-0060-pdn ◽

2014 ◽

Vol 98 (11) ◽

pp. 1580-1580 ◽

Cited By ~ 2

Author(s):

C. Kithan ◽

L. Daiho

Keyword(s):

Leaf Surface ◽

Agricultural Research ◽

Disease Incidence ◽

Indian Agricultural Research Institute ◽

Mountain Range ◽

Pathogenicity Test ◽

Distilled Water ◽

Conidial Suspension ◽

First Report ◽

Initial Symptoms

Etlingera linguiformis (Roxb.) R.M.Sm. of Zingiberaceae family is an important indigenous medicinal and aromatic plant of Nagaland, India, that grows well in warm climates with loamy soil rich in humus (1). The plant rhizome has medicinal benefits in treating sore throats, stomachache, rheumatism, and respiratory complaints, while its essential oil is used in perfumery. A severe disease incidence of leaf blight was observed on the foliar portion of E. linguiformis at the Patkai mountain range of northeast India in September 2012. Initial symptoms of the disease are small brown water soaked flecks appearing on the upper leaf surface with diameter ranging from 0.5 to 3 cm, which later coalesced to form dark brown lesions with a well-defined border. Lesions often merged to form large necrotic areas, covering more than 90% of the leaf surface, which contributed to plant death. The disease significantly reduces the number of functional leaves. As disease progresses, stems and rhizomes were also affected, reducing quality and yield. The diseased leaf tissues were surface sterilized with 0.2% sodium hypochlorite for 2 min followed by rinsing in sterile distilled water and transferred into potato dextrose agar (PDA) medium. After 3 days, the growing tips of the mycelium were transferred to PDA slants and incubated at 25 ± 2°C until conidia formation. Fungal colonies on PDA were dark gray to dark brown, usually zonate; stromata regularly and abundantly formed in culture. Conidia were straight to curved, ellipsoidal, 3-septate, rarely 4-septate, middle cells broad and darker than other two end cells, middle septum not median, smooth, 18 to 32 × 8 to 16 μm (mean 25.15 × 12.10 μm). Conidiophores were terminal and lateral on hyphae and stromata, simple or branched, straight or flexuous, often geniculate, septate, pale brown to brown, smooth, and up to 800 μm thick (2,3). Pathogen identification was performed by the Indian Type Culture Collection, Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi (ITCC Accession No. 7895.10). Further molecular identity of the pathogen was confirmed as Curvularia aeria by PCR amplification and sequencing of the internal transcribed spacer (ITS) regions of the ribosomal DNA by using primers ITS4 and ITS5 (4). The sequence was submitted to GenBank (Accession No. MTCC11875). BLAST analysis of the fungal sequence showed 100% nucleotide similarity with Cochliobolus lunatus and Curvularia aeria. Pathogenicity tests were performed by spraying with an aqueous conidial suspension (1 × 106 conidia /ml) on leaves of three healthy Etlingera plants. Three plants sprayed with sterile distilled water served as controls. The first foliar lesions developed on leaves 7 days after inoculation and after 10 to 12 days, 80% of the leaves were severely infected. Control plants remained healthy. The inoculated leaves developed similar blight symptoms to those observed on naturally infected leaves. C. aeria was re-isolated from the inoculated leaves, thus fulfilling Koch's postulates. The pathogenicity test was repeated twice. To our knowledge, this is the first report of the presence of C. aeria on E. linguiformis. References: (1) M. H. Arafat et al. Pharm. J. 16:33, 2013. (2) M. B. Ellis. Dematiaceous Hyphomycetes. CMI, Kew, Surrey, UK, 1971. (3) K. J. Martin and P. T. Rygiewicz. BMC Microbiol. 5:28, 2005. (4) C. V. Suberamanian. Proc. Indian Acad. Sci. 38:27, 1955.

Download Full-text

First Report of Anthracnose Caused by Colletotrichum fragariae on Cyclamen in North Carolina

Plant Disease ◽

10.1094/pdis-06-11-0475 ◽

2011 ◽

Vol 95 (11) ◽

pp. 1480-1480 ◽

Cited By ~ 3

Author(s):

B. Liu ◽

M. Munster ◽

C. Johnson ◽

F. J. Louws

Keyword(s):

North Carolina ◽

Sequence Analysis ◽

Room Temperature ◽

Plant Diseases ◽

Pathogenicity Test ◽

Conidial Suspension ◽

Disease Symptoms ◽

First Report ◽

Leaf Spots ◽

Colletotrichum Fragariae

In November 2009, cyclamen (Cyclamen persicum) plants with disease symptoms from a commercial greenhouse operation in the western part of North Carolina were sent to the Plant Diseases and Insect Clinic at North Carolina State University. Symptoms consisted of coalescing reddish and tan necrotic leaf spots with concentric circles. Other symptoms included darkened vascular tissue and decay of the corm, large roots, and petioles. Diseased leaves and stems were surface sterilized in 0.5% sodium hypochlorite for 3 min, air dried, and placed in petri dishes containing alkaline water agar. After 3 days of incubation at room temperature, fungal colonies were transferred to acidified potato dextrose agar. Isolation frequency after 5 days was 33% (three of nine pieces) and 16% (one of six pieces) from small leaf spots and petioles, respectively. Pure cultures of isolates were gray and black with abundant, aerial, gray whitish mycelia. Diseased plants were also incubated in a moist chamber at room temperature and sporulation was observed within 7 days. Conidia were tapered with rounded ends and produced in the acervulus and on the tips of setae, which is consistent with the morphology of described isolates of Colletotrichum fragariae. Similar setae were also observed directly on the fine roots of the original sample. The pathogenicity of single-spore cultures was tested by spraying four 2-month-old cyclamen plants with a conidial suspension (106 conidia/ml) and the plants were kept in a humid chamber for 24 h. Noninoculated controls (four plants) were sprayed with distilled water and subjected to the same conditions. The pathogenicity test was also repeated. Inoculated plants and controls were placed in a greenhouse with a temperature range from 22 to 25°C. After 7 to 10 days, symptomatic leaves and stems were observed on all the inoculated plants but not on the control plants. Fungi reisolated from 10 symptomatic leaf tissues had identical morphological features as the original isolates. Fungal DNA was extracted with DNeasy Plant Mini DNA Extraction Kits following the manufacturer's protocol (Qiagen Inc., Valencia, CA). Sequence analysis of the rRNA internal transcribed spacer (ITS) region of the cyclamen isolate (GenBank Accession No. HQ188923), based on the fragment amplified with ITS1 and ITS4 primers, showed 100% similarity to isolates of C. fragariae deposited in GenBank (Accession Nos. FJ172290 [ATCC MYA-4443 from cyclamen] and FJ810510 [ATCC MYA-4442 from silver date palm]) and Florida isolate C16 isolated from strawberry (1). In addition, the morphology and ITS sequences of the cyclamen isolate were identical to those of the C. fragariae voucher isolate from strawberry (GU174546). Results from disease symptoms, colony and spore morphology, pathogenicity tests, and ITS sequence analysis suggest that C. fragariae was the pathogen responsible for the disease symptoms on cyclamens. To our knowledge, this is the first report of a disease caused by C. fragariae on cyclamen in North Carolina and complements an earlier report from Florida (1). Reference: (1) S. J. MacKenzie et al. Plant Dis. 92:1432, 2008.

Download Full-text

First Report of Fusarium proliferatum Causing Fruit Rot of Grapes (Vitis vinifera) in Pakistan

International Journal of Phytopathology ◽

10.33687/phytopath.007.02.2589 ◽

2018 ◽

Vol 7 (2) ◽

pp. 85-88 ◽

Cited By ~ 1

Author(s):

Salman Ghuffar ◽

Gulshan Irshad ◽

Fengyan Zhai ◽

Asif Aziz ◽

Hafiz M. Asadullah M. Asadullah ◽

...

Keyword(s):

Vitis Vinifera ◽

Negative Control ◽

Fusarium Proliferatum ◽

Fruit Rot ◽

Pathogenicity Test ◽

Distilled Water ◽

Conidial Suspension ◽

Fruit Crop ◽

First Report ◽

Thin Walled

Grapes (Vitis vinifera) are the important fruit crop in Pakistan, mostly cultivated for edible purpose. In September 2016, unusual fruit rot symptoms were observed 3-5 days after harvesting on grapes cv. Kishmishi in post-harvest packing houses in Jehlum district (32°56'22.3"N 73°43'31.4"E) of Punjab province. To determine the disease incidence, a total of 10 boxes of grapes from 5 different locations were selected randomly. Each box contained average 12 bunches and 30 bunches out of 120 inspected bunches displayed typical symptoms of the disease. The initial Symptoms were small, round, water-soaked lesions that rapidly developed into soft, white to light pink mycelium near the centre of infected fruits (Figure 1). A total of 186 symptomatic berries were surface sterilized with 1% sodium hypochlorite, rinsed three times with sterile distilled water and dried by placing on filter paper for 45 sec. Sterilized tissues (approximately 4 mm3) were excised and incubated on potato dextrose agar (PDA) medium at 25 ± 4°C. One week after incubation, colonies with abundant aerial mycelium were initially white, cottony and turned to violet and dark purple with age (Figure 2). A total of 25 isolates were examined morphologically. Macroconidia were slender, thin-walled, 3 to 5 septate, curved apical cell, with 20.9 to 45.2 × 3.2 to 7.1 μm and Microconidia were thin-walled, aseptate, club-shaped with 4.5 to 11.2 × 2.3 to 4.1 μm (Figure 3). These characteristics best fit for the description of Fusarium proliferatum (Leslie and Summerell, 2006). Portions of the internal transcribed spacer (ITS) region were sequenced (White et al., 1990). Sequences of two isolates Fus 07 and Fus 09 (GenBank Accessions; MH444366 and MH464139) showed 100% identity to the corresponding gene sequences of Fusarium proliferatum (GenBank Accessions; MH368119, MF033172 and KU939071) (Figure 4). Pathogenicity test was performed by inoculation with 50-μl conidial suspension (1 × 106conidia/ml) of two isolates onto three non-wounded and four wounded asymptomatic grapes berries. Sterile distilled water was used for a negative control (Figure 5). The experiment was conducted twice and berries were incubated at 25 ± 2°C in sterile moisture chambers (Ghuffar et al., 2018). White to light pink mycelium in appearance with the original symptoms were observed on both wounded and non-wounded inoculated berries after 3 days, whereas no symptoms were observed on the negative control. The morphology of the fungus that was re-isolated from each of the inoculated berries was identical to that of the original cultures. Fusarium proliferatum, one of the destructive species, causes diseases like foot-rot of corn (Farr et al., 1990), root rot of soybean (Díaz Arias et al., 2011), bakanae of rice (Zainudin et al., 2008), wilt of date palm (Khudhair et al., 2014), tomato wilt (Chehri, 2016) and tomato fruit rot (Murad et al., 2016). To our knowledge, this is the first report of Fusarium proliferatum causing fruit rot of grapes in Pakistan, where the disease poses a significant threat to the sustainability of this major fruit crop.

Download Full-text

First Report of Fusarium Wilt in Blueberry (Vaccinium corymbosum) Caused by Fusarium oxysporum in China

Plant Disease ◽

10.1094/pdis-02-14-0167-pdn ◽

2014 ◽

Vol 98 (8) ◽

pp. 1158-1158 ◽

Cited By ~ 1

Author(s):

Y. H. Liu ◽

T. Lin ◽

C. S. Ye ◽

C. Q. Zhang

Keyword(s):

Fusarium Oxysporum ◽

Infected Plant ◽

Morphological Characteristics ◽

Vaccinium Corymbosum ◽

Morphological Characterization ◽

Internal Transcribed Spacers ◽

Fluorescent Light ◽

Pathogenicity Test ◽

Conidial Suspension ◽

First Report

Blueberry (Vaccinium corymbosum) production is developing quickly in China with about 20,000 ha presently cultivated. In 2010 in Lin'an, Zhejiang Province, plants developed an apparently new disease of blueberry (cv. Duke) with symptoms consisting of wilting of foliage, stunting of plants, and reduced fruit yields. Internal vascular and cortical tissues of plant crowns showed a brown to orange discoloration. Approximately 3% of the plants in the commercial plantings were affected and eventually died after 50 to 60 days. Infected plant samples (stems and roots) collected from different fields were surface sterilized with 1.5% sodium hypochlorite for 2 min, rinsed in water, plated on 2% potato dextrose agar (PDA), and incubated at 25°C in the dark for 1 week. Single conidium cultures were consistently isolated and cultured on acidified PDA (APDA) for morphological characterization (1,2). Colonies were light with purple mycelia, and beige or orange reverse colony colors developed after 7 days incubation at 25°C. Colonies producing abundant microconidia and macroconidia. Microconidia were hyaline and oval-ellipsoid to cylindrical (3.9 to 9.6 × 1.1 to 3.4 μm). Macroconidia were 3 to 5 septate and fusoid-subulate with a pedicellate base (28.6 to 37.5 × 3.3 to 4.2 μm). Morphology and development of macroconidia and microconida were consistent with a description of Fusarium oxysporum Schltdl (1,2). The ribosomal internal transcribed spacers ITS1 and ITS2 of eight isolates were amplified using primers ITS1/ITS4 on DNA extracted from mycelium and nucleotide sequences showed 100% similarity to that of F. oxysporum. To confirm pathogenicity, 20 blueberry plants (cv. Duke) were inoculated by dipping the roots into a conidial suspension (107 conidia per ml) for 30 min. The inoculated plants were transplanted into pots containing sterilized peat and maintained at 25°C and 100% relative humidity in a growth chamber with a daily 12-h photoperiod of fluorescent light. The pathogenicity test was conducted twice. Within 40 days, all inoculated plants developed wilt symptoms similar to that observed in the field. No symptoms were observed on plants dipped into distilled water. The fungus was successfully re-isolated from crowns and roots cultured on APDA, exhibiting morphological characteristics identical to F. oxysporum (1,2), confirming Koch's postulates. To our knowledge, this is the first report of blueberry wilt caused by Fusarium. References: (1) P. M. Kirk et al. The Dictionary of the Fungi, 10th edition, page 159. CABI Bioscience, Wallingford, UK, 2008. (2) W. C. Snyder and H. N. Hansen. Am. J. Bot. 27:64, 1940.

Download Full-text

First Report of Fusarium Wilt Caused by Fusarium oxysporum on Strawberry in Spain

Plant Disease ◽

10.1094/pdis-93-3-0323b ◽

2009 ◽

Vol 93 (3) ◽

pp. 323-323 ◽

Cited By ~ 22

Author(s):

F. T. Arroyo ◽

Y. Llergo ◽

A. Aguado ◽

F. Romero

Keyword(s):

Fusarium Oxysporum ◽

Fusarium Wilt ◽

Potato Dextrose Agar ◽

Fluorescent Light ◽

Pathogenicity Test ◽

Conidial Suspension ◽

Soilless Culture ◽

Fragaria Ananassa ◽

Pcr Assay ◽

First Report

In the spring of 2007, wilted and dead strawberry plants (Fragaria × ananassa Duch. cvs. Camarosa and Ventana) were observed in a soilless culture system in Huelva, southwestern Spain. Approximately 8% of the plants in the field died. Isolations from necrotic crowns and roots and necrotic flowers were made on potato dextrose agar after disinfestation in 0.6% NaOCl for 30 s. Colonies with light purple mycelia and beige or orange reverse colony colors developed after 9 days of incubation at 25°C. Colonies produced abundant microconidia, macroconidia, and chlamydospores. Microconidia were hyaline and oval-ellipsoid to cylindrical (5.9 to 9.2 × 2.1 to 3.4 μm). Macroconidia were 3 to 5 septate and fusoid-subulate with a pedicellate base (28.8 to 37.3 × 3.2 to 4.3 μm). Morphology and growth matched descriptions of Fusarium oxysporum Schlechtend emend. Snyder & Hansen (2). A PCR assay for amplification of r-DNA using primers PFO2 and PFO3 established the identity of the isolate as F. oxysporum (1). To confirm the pathogenicity of the fungus, roots of 30-day-old strawberry cvs. Camarosa and Ventana (20 plants each) were inoculated by dipping the roots into a conidial suspension (107 conidia per ml) for 15 min. The inoculated plants were transplanted into plastic pots containing sterilized peat and maintained at 25°C and 100% relative humidity in a growth chamber with a daily 12-h photoperiod of fluorescent light. The pathogenicity test was conducted twice. Within 30 days, all inoculated plants developed wilt symptoms similar to that observed in the field and eventually 75% of the plants died. No symptoms were observed on plants dipped in distilled water. The fungus was successfully reisolated from crowns, roots, and necrotic flowers, fulfilling Koch's postulates. To our knowledge, this is the first report of the occurrence of Fusarium wilt caused by F. oxysporum on strawberry plants in Spain. References: (1) V. Edel et al. Mycol. Res. 104:518, 2000. (2) W. C. Snyder and H. N. Hansen. Am. J. Bot. 27:64, 1940.

Download Full-text