scholarly journals Occurrence of Grapevine Trunk Disease Caused by Botryosphaeria rhodina in China

Plant Disease ◽  
2011 ◽  
Vol 95 (2) ◽  
pp. 219-219 ◽  
Author(s):  
J.-Y. Yan ◽  
X.-H. Li ◽  
F.-F. Kong ◽  
Z.-Y. Wang ◽  
L.-Z. Gong ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
Early Summer ◽  
Academic Press ◽  
Single Spore ◽  
Rdna Its ◽  
Agar Plug ◽  
Pcr Products ◽  
Botryosphaeria Rhodina ◽  
Causal Pathogen ◽  
Cork Borer

In the early summer of 2009, grapevine (Vitis vinifera), an important fruit crop in China, declined in most of the vineyards in Hunan, Hubei, and Zhejiang provinces. Characteristic symptoms of Botryosphaeria canker were apparent, including trunk cankers (visible in cross-section), leaf drop, shriveling and drying of fruit clusters, and berry rot (1). To identify the causal pathogen, we tested 126 samples by attempting to culture the pathogen from a small piece of tissue from the canker margin between the necrotic and apparently healthy tissue. Plant tissue was surface sterilized by placing it in 75% ethanol for 1 min and rinsed with sterilized water three times before culturing to potato dextrose agar (PDA) at 28°C. Five days later, the cultures were hyphal-tip purified and then single-spore isolates were used for identification. On the basis of colony characteristics in PDA, these colonies were identified as Botryosphaeria spp. (2). They were grayish white, becoming dark brown with age, and pycnidia were formed after incubation for approximately 9 days. Conidia measured 11 to 15 × 22 to 28 μm. A subset of isolates were used for rDNA ITS (internal transcribed spacer) sequence analysis with primers ITS1 and ITS4 (3). PCR products were separated by electrophoresis and bands were purified (Qiagen Plasmid Mini Kit; Qiagen, Valencia, CA) for sequencing (Sunbiotech Company, Beijing). BLAST searches of three ITS sequences (Accession Nos. GU226851, GU226853, and GU226856) had 100% identity to B. rhodina. EF1-α and β-tubulin sequence analysis gave similar results. Koch's postulates were completed in the laboratory on grape shoots inoculated with two isolates of B. rhodina, originally isolated from plants in the field with symptoms of Botryosphaeria canker. Isolates were incubated on PDA at 25°C for 1 week. Inoculations were made on green shoots of V. vinifera cvs. Muscat Hamburg and Crimson Seedless. Five shoots per cultivar were inoculated per isolate by wounding with a 4-mm cork borer (2 mm deep), placing a colonized agar plug on the wound, and wrapping the wound with Parafilm. Controls were mock inoculated with an agar plug from sterile PDA. Inoculated shoots were incubated in the laboratory in the dark under moist conditions for 10 days at 25°C. Inoculated shoots had necrotic cankers after 10 days and B. rhodina was recovered from each canker margin. The results suggest that some grapevines in China with symptoms of Botryosphaeria canker were indeed infected by B. rhodina. To our knowledge, this is the first report of this pathogen on grapevine in China. References: (1) J. Luque et al. Mycologia 97:1111, 2005. (2) J. M. Niekerk et al. Mycologia 96:781, 2004. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

scholarly journals First Report of Grapevine Trunk Disease Caused by Botryosphaeria obtusa in China

Plant Disease ◽  
2011 ◽  
Vol 95 (5) ◽  
pp. 616-616 ◽  
Author(s):  
J.-Y. Yan ◽  
Y.-L. Peng ◽  
Y. Xie ◽  
X.-H. Li ◽  
S.-W. Yao ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
First Report ◽  
Trunk Disease ◽  
Agar Plug ◽  
Brown Discoloration ◽  
Its Sequence Analysis ◽  
Pcr Products ◽  
Pure Cultures ◽  
Botryosphaeria Obtusa ◽  
Causal Pathogen

In September 2010, grapevine (Vitis vinifera) trunk diseases were observed in several vineyards of Yantai District in Shandong Provinces and Changli County of Hebei Provinces of China. Characteristic symptoms of Botryosphaeria canker were apparent, including dark brown discoloration on the trunk (visible in cross-section), cob base shriveling, drying of fruit clusters, and berry falling (2). To identify the causal pathogen, culturing of fungi was attempted from 387 small pieces of tissue from the canker margins of 43 diseased plants. Samples were surface disinfected by placing them in 75% ethanol for 1 min and rinsing with sterilized water three times before culturing on potato dextrose agar (PDA) at 28°C for 7 to 10 days. Fungi isolated were single spored to obtain pure cultures. On the basis of colony characteristics on PDA, 18 isolates from the 387 tissue pieces were eventually identified as Botryosphaeria obtusa (1), Most of the other fungi isolated were B. dothidea. B. obtusa colonies were grayish white, becoming dark brown with age, and pycnidia were formed after incubation for approximately 7 days. Conidia measured 8 to 11 × 17 to 26 μm (n= 50). Two isolates were used for rDNA internal transcribed spacer (ITS) sequence analysis with primers ITS1 and ITS4 (3). PCR products were separated by electrophoresis and bands were purified for legation with PMD-18T (Takara Company, Dalian, China) vector for sequencing. BLAST searches of two ITS sequences had 99 to 100% identity to B. obtusa. EF1-α and β-tubulin sequence analysis gave similar results. Koch's postulates were completed in the greenhouse on grape shoots inoculated with two isolates of B. obtusa originally isolated from diseased plants in the field. Inoculations were made on green shoots of V. vinifera cv. Dunkelfelder T. Six shoots were inoculated per isolate by wounding with a 4-mm cork borer (2 mm deep) and placing a colonized agar plug from a 5-day-old culture on the wound and wrapping it with Parafilm. Controls were mock inoculated with an agar plug from sterile PDA. Inoculated shoots were incubated in the dark under moist conditions in the laboratory for 8 to 10 days at 25°C. Inoculated shoots had necrotic cankers after 8 to 10 days and B. obtusa was recovered from each canker margin. The results indicated that some grapevines in China with symptoms of Botryosphaeria canker were infected by B. obtusa. To our knowledge, this is the first report of this pathogen causing trunk disease on grapevine in China. References: (1) A. Taylor et al. Australas. Plant Pathol. 34:187, 2005. (2) J. R. Úrbez-Torres et al. Plant Dis. 92:519, 2008. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

scholarly journals First Report of Brown Rot of Apricot Caused by Monilia mumecola

Plant Disease ◽  
2014 ◽  
Vol 98 (5) ◽  
pp. 694-694 ◽  
Author(s):  
L. F. Yin ◽  
S. N. Chen ◽  
M. L. Cai ◽  
G. Q. Li ◽  
C. X. Luo
Keyword(s):  
Brown Rot ◽  
Prunus Armeniaca ◽  
Its Sequences ◽  
Academic Press ◽  
Single Spore ◽  
First Report ◽  
Blast Search ◽  
Mean Size ◽  
Cork Borer ◽  
Germ Tubes

In May 2013, apricot (Prunus armeniaca) fruits covered with grayish, conidial masses were collected from an unknown cultivar in an experimental field of Huazhong Agricultural University, Wuhan, Hubei Province. About 3 to 5% of fruit was infected and affected apricots had tan to white zones of sporulation, which resembled brown rot caused by Monilia species. Conidia were harvested from the surface of the sporulating apricot fruit and spread onto shallow potato dextrose agar (PDA) media (about 2 mm in thickness) using sterile cotton swabs. Conidia were lemon-shaped and mean size was 15.7 (10 to 22.5) × 25 (16.25 to 35) μm. Conidia on PDA were incubated at 23°C for 3 h in darkness, then observed under microscope. More than two germ tubes were produced from each conidium, which was the distinctive trait of Monilia mumecola species (2). Single-spore isolates were obtained and 3 isolates were cultured on PDA in petri dishes. Mycelium grew at an average of 15 mm per day, and the colony showed concentric rings of mycelium with lobbed margins at 23°C in darkness. A 712-bp fragment was PCR amplified from β-tubulin gene (TUB2) of all the nine isolates investigated indicative of M. mumecola (2). The ribosomal ITS1-5.8S-ITS2 regions of nine isolates were also PCR-amplified from genomic DNA using primers ITS1 and ITS4 and then sequenced (4). ITS sequences were identical to ITS sequences of M. mumecola from China (HQ908786) and Japan (AB125613, AB125614, and AB125620), but only has 98% and 97% identity with the closest species M. laxa (EU042149) and M. fructicola (HQ908789) according to BLAST search in GenBank. Pathogenicity was confirmed by inoculating mycelial plugs of three isolates into six surface-sterilized apricots wounded with a 6-mm diameter sterile cork borer. Control fruit received plain PDA plugs and was incubated in a moist chamber at 23°C with 12 h light/12 h dark. All inoculated fruit developed typical brown rot symptoms with sporulating areas as described above after 3 days of incubation, while control fruits remained healthy. The developing spores on inoculated fruit were re-isolated and confirmed to be M. mumecola. M. mumecola was first isolated from Prunus mume in Japan in 1982 as an unknown Monilia species (3), then identified and the nomenclature was provided in 2004 (1). To our knowledge, this is the first report of M. mumecola on P. armeniaca indicating that M. mumecola has spread to different hosts. References: (1) Y. Harada et al. J. Gen. Plant Pathol. 70:297, 2004. (2) M. J. Hu et al. Plos One, 6(9):e24990, 2011. (3) S. Nakao. Kongetsu-no-noyaku, 1:92, 1992. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, 1990.

Mitochondrial Cytochrome b DNA Sequence Variations: An Approach to Fish Species Identification in Processed Fish Products

2005 ◽  
Vol 68 (2) ◽  
pp. 421-425 ◽  
Author(s):  
TIZIANA PEPE ◽  
MICHELE TROTTA ◽  
ISOLINA DI MARCO ◽  
PAOLA CENNAMO ◽  
ANIELLO ANASTASIO ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
Cytochrome B ◽  
Fish Species ◽  
Mitochondrial Cytochrome ◽  
Fish Products ◽  
Community Regulation ◽  
Sequence Variations ◽  
Fish Species Identification ◽  
Mitochondrial Cytochrome B ◽  
Pcr Products

The identification of fish species in food products is problematic because morphological features of the fish are partially or completely lost during processing. It is important to determine fish origin because of the increasing international seafood trade and because European Community Regulation 104/2000 requires that the products be labeled correctly. Sequence analysis of PCR products from a conserved region of the cytochrome b gene was used to identity fish species belonging to the families Gadidae and Merluccidae in 18 different processed fish products. This method allowed the identification of fish species in all samples. Fish in all of the examined products belonged to these two families, with the exception of one sample of smoked baccalà (salt cod), which was not included in the Gadidae cluster.

scholarly journals First Report of Colletotrichum siamense Causing Anthracnose of Monstera deliciosa in Zhanjiang, China

Plant Disease ◽  
2020 ◽  
Author(s):  
Yue Lian Liu ◽  
Jian Rong Tang ◽  
Yu Han Zhou
Keyword(s):  
Disease Incidence ◽  
Morphological Characteristics ◽  
Pathogenicity Test ◽  
Sterile Water ◽  
Single Spore ◽  
First Report ◽  
Rdna Its ◽  
Colletotrichum Siamense ◽  
Monstera Deliciosa ◽  
Pure Cultures

Monstera deliciosa Liebm is an ornamental foliage plant (Zhen et al. 2020De Lojo and De Benedetto 2014). In July of 2019, anthracnose lesions were observed on leaves of M. deliciosa cv. Duokong with 20% disease incidence of 100 plants at Guangdong Ocean University campus (21.17N,110.18E), Guangdong Province, China. Initially affected leaves showed chlorotic spots, which coalesced into larger irregular or circular lesions. The centers of spots were gray with a brown border surrounded by a yellow halo (Supplementary figure 1). Twenty diseased leaves were collected for pathogen isolation. Margins of diseased tissue was cut into 2 × 2 mm pieces, surface-disinfected with 75% ethanol for 30 s and 2% sodium hypochlorite (NaOCl) for 60 s, rinsed three times with sterile water before isolation. Potato dextrose agar (PDA) was used to culture pathogens at 28℃ in dark. Successively, pure cultures were obtained by transferring hyphal tips to new PDA plates. Fourteen isolates were obtained from 20 leaves. Three single-spore isolates (PSC-1, PSC-2, and PSC-3) were obtained ,obtained, which were identical in morphology and molecular analysis (ITS). Therefore, the representative isolate PSC-1 was used for further study. The culture of isolate PSC-1 on PDA was initially white and later became cottony, light gray in 4 days, at 28 °C. Conidia were single celled, hyaline, cylindrical, clavate, and measured 13.2 to 18.3 µm × 3.3 to 6.5 µm (n = 30). Appressoria were elliptical or subglobose, dark brown, and ranged from 6.3 to 9.5 µm × 5.7 to 6.5 µm (n = 30). Morphological characteristics of isolate PSC-1 were consistent with the description of Colletotrichum siamense (Prihastuti et al. 2009; Sharma et al. 2013). DNA of the isolate PSC-1 was extracted for PCR sequencing using primers for the rDNA ITS (ITS1/ITS4), GAPDH (GDF1/GDR1), ACT (ACT-512F/ACT-783R), CAL (CL1C/CL2C), and TUB2 (βT2a/βT2b) (Weir et al. 2012). Analysis of the ITS (accession no. MN243535), GAPDH (MN243538), ACT (MN512640), CAL (MT163731), and TUB2 (MN512643) sequences revealed a 97-100% identity with the corresponding ITS (JX010161), GAPDH (JX010002), ACT (FJ907423), CAL (JX009714) and TUB2 (KP703502) sequences of C. siamense in GenBank. A phylogenetic tree was generated based on the concatenated sequences of ITS, GAPDH, ACT, CAL, and TUB2 which clustered the isolate PSC-1 with C. siamense the type strain ICMP 18578 (Supplementary figure 2). Based on morphological characteristics and phylogenetic analysis, the isolate PSC-1 associated with anthracnose of M. deliciosa was identified as C. siamense. Pathogenicity test was performed in a greenhouse at 24 to 30oC with 80% relative humidity. Ten healthy plants of cv. Duokong (3-month-old) were grown in pots with one plant in each pot. Five plants were inoculated by spraying a spore suspension (105 spores ml-1) of the isolate PSC-1 onto leaves until runoff, and five plants were sprayed with sterile water as controls. The test was conducted three times. Anthracnose lesions as earlier were observed on the leaves after two weeks, whereas control plants remained symptomless. The pathogen re-isolated from all inoculated leaves was identical to the isolate PSC-1 by morphology and ITS analysis, but not from control plants. C. gloeosporioides has been reported to cause anthracnose of M. deliciosa (Katakam, et al. 2017). To the best of our knowledge, this is the first report of C. siamense causing anthracnose on M. deliciosa in ChinaC. siamense causes anthracnose on a variety of plant hosts, but not including M. deliciosa (Yanan, et al. 2019). To the best of our knowledge, this is the first report of C. siamense causing anthracnose on M. deliciosa, which provides a basis for focusing on the management of the disease in future.

scholarly journals First Report of Curvularia Leaf Blight on Curcuma wenyujin Caused by Curvularia clavata in China

Plant Disease ◽  
2013 ◽  
Vol 97 (1) ◽  
pp. 138-138 ◽  
Author(s):  
X. Y. Chen ◽  
J. D. Feng ◽  
Z. Su ◽  
C. Sui ◽  
X. Huang
Keyword(s):  
Morphological Characteristics ◽  
Its Region ◽  
Leaf Blight ◽  
Hainan Province ◽  
Continuous Cropping ◽  
Single Spore ◽  
Chinese Medicinal Herb ◽  
First Report ◽  
Causal Pathogen ◽  
Blight Disease

Curcuma wenyujin Y.H. Chen & C. Ling is a traditional Chinese medicinal herb in the Zingiberaceae family. Commonly known as Wen yujin, the root is widely used for alleviating pain and protecting the liver. A severe leaf blight disease was observed in three C. wenyujin farms in Hainan Province of China in October 2010. The obvious symptoms of leaf blight, yellow to brown irregular lesions (1 to 20 cm) on C. wenyujin, usually began at the tips of leaves and the main veins. This disease, especially severe from August to October, caused heavy damage and 100% of mature plants (10 months old) in farms were infected. The disease was most severe when continuous cropping was performed and showed slight improvement when rotation was adopted. Farmers usually sprayed carbendazim (50% WP) and thiophanate-methyl (70% WP) to control this disease, but these treatments were not effective. To isolate the causal pathogen, diseased plants were collected in October 2010 from a field of the Hainan Branch Institute of Medicinal Plant Development in Hainan Province. Lesion tissue was removed from the border between symptomatic and healthy tissue, surface sterilized in 75% ethanol for 1 min, washed in three changes of sterile distilled water, transferred to potato dextrose agar (PDA) plates, and incubated at 28°C for 7 days. Single spore cultures of five isolates were obtained and identified as Curvularia clavata based on morphological characteristics (1). Conidia measured 20 to 29 × 7.5 to 10.5 μm (n = 100), were curved, 3-septate, and the third cell from the base was larger and darker than the others. Mycelia of single spore cultures growing on PDA for 5 days were used for DNA extraction using a plant genomic DNA kit (TIANGEN, Beijing). The internal transcribed spacer (ITS) region of the rDNA was amplified using primers ITS1 and ITS4. The amplicons were 562 bp in length (GenBank Accession No. JQ730852) and had 99% nucleotide identity with the GenBank Accession No. JN021115 and AF071336 of C. clavata. Pathogenicity tests were conducted using fresh and healthy detached Curcuma wenyujin leaves. Mycelial discs (10 mm) removed from a 5-day-old colony on PDA were used for inoculation. Each isolate was inoculated on three distinct leaves (two distinct inoculations per leaf). Three additional leaves inoculated with sterile PDA discs were used as control. Inoculated leaves were covered with a polythene film to maintain high humidity. Leaves in trays were kept in a growth chamber at 28°C and observed for symptom appearance every day. Five days after inoculation, inoculated leaves developed blight symptoms similar to those observed on naturally infected leaves. No symptoms were observed on non-inoculated leaves. C. clavata was reisolated from the inoculated leaves, thus fulfilling Koch's postulates. C. clavata has been previously reported to be economically important on a number of other hosts (2). To our knowledge, this is the first report of Curvularia leaf blight on Curcuma wenyujin caused by C. clavata in China. References: (1) A. M Mandokhot et al. Eur. J. Plant Pathol.78:65, 1972. (2) T. Y. Zhang et al. Flora fungorum sinicorum: Beijing, China, 2010.

scholarly journals Analysis of Genetic and Molecular Identity Among Field Isolates of the Rice Blast Fungus with an International Differential System, Rep-PCR, and DNA Sequencing

Plant Disease ◽  
2013 ◽  
Vol 97 (4) ◽  
pp. 491-495 ◽  
Author(s):  
Junjie Xing ◽  
Yulin Jia ◽  
James C. Correll ◽  
Fleet N. Lee ◽  
Richard Cartwright ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
Dna Sequencing ◽  
Differential System ◽  
Repetitive Element ◽  
Chain Reaction ◽  
Specific Primers ◽  
Molecular Identity ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Avr Gene

The Pi-ta gene deployed in southern U.S. rice germplasm is effective in preventing the infection by strains of Magnaporthe oryzae isolates that carry the avirulence (AVR) gene AVR-Pita1. In the present study, 169 isolates from rice (Oryza sativa) cultivars, with and without Pi-ta, were analyzed for their genetic identity using an international differential system, repetitive element-based polymerase chain reaction (Rep-PCR), and sequence analysis of PCR products of AVR-Pita1. These isolates belong to the races IA1, IB1, IB17, IC1, and IC17 of M. oryzae. These isolates were further classified into 15 distinct groups by Rep-PCR. There was a predominant group within each race. Pathogenicity assays on ‘Katy’ (Pi-ta) and ‘M202’ (pi-ta) rice determined that IC1 was virulent to Katy and M202; IB17, IC17, and most of IA1 and IB1 were avirulent to Katy and virulent to M202, suggesting that the Pi-ta gene in Katy is responsible for preventing infection by these isolates. Consistently, AVR-Pita1 was not amplified from 28 virulent isolates. One AVR-Pita1 allele was amplified by AVR-Pita1-specific primers in 78 avirulent isolates. Interestingly, different AVR-Pita1 alleles were found in each of the 12 avirulent isolates, as determined by DNA sequencing. Sequence analysis of 90 PCR products revealed 10 AVR-Pita1 haplotypes, 4 of which were new. In total, 12 amino acid changes were identified in the new variants when compared with the first described AVR-Pita sequence (AF207841). The finding of isolates with altered AVR-Pita1 from rice cultivars with and without Pi-ta suggests that these virulent isolates were adapted to the field environments in the southern United States. Further research will be needed to verify this prediction.

scholarly journals Deteksi dan analisis sekuen gen inhibitor proteinase pada beberapa klon kakao harapan tahan penggerek buah kakao dari Sulawesi Selatan Detection and sequence analysis of proteinase inhibitor gene in cacao clones putatively cacao pod borer-tolerant from South Sulawesi

2016 ◽  
Vol 72 (1) ◽  
Author(s):  
Abdul Mollah S. JAYA ◽  
Hajrial ASWIDINNOOR ◽  
Djoko SANTOSO
Keyword(s):  
Sequence Analysis ◽  
Proteinase Inhibitor ◽  
Genomic Dna ◽  
Genetic Similarity ◽  
Sequence Analyses ◽  
South Sulawesi ◽  
Pod Borer ◽  
Special Programs ◽  
Pcr Products ◽  
Alignment Analysis

Summary Cacao is socially and economically an important commodity for Indonesia, in which the cacao plantations have been challenged with a threatening pest, cacao pod borer (CPB). This research aimed to identify and clone PIN (proteinase inhibitor), a gene carrying resistance of plant to some chewing pests like CPB. The methodology included several experiments. Detection of PIN in cacao was done by PCR using PIN-specific heterologous primers and cacao genomic DNA as templates. Cloning vector pGEM-T was utilized to clone the PCR products. Sequence analysis was conducted with BlastX and Blast Special programs from NCBI. Alignment analysis to determine genetic similarity was performed with ClustalW from EBI. Thirteen of the 18 clones tested were detected to have PIN homologs. Two DNA fragments from cacao clones putatively tolerant to CPB, MJ-1 and LW-1, were sequenced. One of them, MJ-1 was cloned. Sequence analyses of the fragments of both cacao clones, indicated that they have PIN homologs and a very closed genetic relation with 96% level of similarity. Ringkasan Kakao adalah komoditas yang secara sosial maupun ekonomi penting bagi Indonesia, dimana perkebunan kakao menghadapi masalah serius hamapenggerek buah kakao (PBK). Penelitian ini bertujuan mengidentifikasi dan mengklon PIN (inhibitor proteinase), gen yang membawa sifat ketahanan tanaman terhadap hama ulat seperti PBK. Metodologinya terdiri dari beberapa percobaan. Deteksi PIN di dalam kakao dikerjakan dengan PCR menggunakan primer heterologous yang spesifik terhadap PIN dan DNA genomik kakao sebagai templetnya. Vektor kloning pGEM-T digunakan untuk mengklon produk PCR. Analisis sekuen dilakukan dengan program BlastX dan Blast spesial dari NCBI. Analisis penjajaran (alignment) untuk menentukan kemiripan genetik menggunakan program ClustalW dari EBI. Tiga belas dari 18 klon kakao yang diuji  menunjukkan adanya  homolog  PIN. Dua DNA fragmen dari klon harapan tahan, MJ-1 dan LW-1, telah ditentukan sekuen nukleotidanya. Satu diantara-nya, MJ-1 berhasil diklon. Analisis sekuen  kedua klon tersebut menunjukkan identitas sebagai homolog PIN dan keduanya memiliki kemiripan genetik yang tinggi.

scholarly journals Leaf Spot of Arugula, Caused by Alternaria japonica, in California

Plant Disease ◽  
2014 ◽  
Vol 98 (9) ◽  
pp. 1272-1272 ◽  
Author(s):  
T. E. Tidwell ◽  
C. L. Blomquist ◽  
S. Rooney-Latham ◽  
H. J. Scheck
Keyword(s):  
Its Region ◽  
The United States ◽  
Green Leaf ◽  
Academic Press ◽  
Leaf Spots ◽  
Agar Plug ◽  
Half Strength ◽  
Dark Green ◽  
Hydroponically Grown ◽  
Dark Green Leaf

Arugula (Eruca vesicaria subsp. sativa (Mill.) Thell. is a Cruciferous plant used for culinary purposes. From 2012 to 2013, a foliar disease seriously impacted the growth and quality of about 0.1 ha of hydroponically grown arugula at a Santa Barbara County nursery. Samples of affected arugula seedlings exhibited adaxial and abaxial symptoms of mottling with circular to oval, water soaked, dark green leaf spots, each 1 to 3 mm in diameter, and some of which coalesced. Conidia of an Alternaria sp. were observed on the foliage. Symptomatic leaf pieces were disinfested with 0.6% NaOCl, blotted dry, and plated on acidified potato dextrose agar (APDA). Cultures were incubated under near-UV lights for 24 h/day. Olivaceous-grey colonies of the same Alternaria species observed on the leaves grew after 7 days. After 21 days on carrot-piece agar (3), the fungus produced beakless conidia with longitudinal and constricted transverse septa that measured 30.0 to 69.0 × 12.5 to 20.0 μm and were borne singly or in short chains of 2 to 3 conidia. In addition, knots of dark, thick-walled micro-chlamydospores were produced by the hyphae. The fungus was identified morphologically as Alternaria japonica Yoshii (2), and the species confirmed by sequence analysis. A portion of the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) was amplified using ITS1 and ITS4 primers (4). The sequence (GenBank Accession No. KJ126846) was 100% identical to the ITS rDNA sequence of an isolate of A. japonica (KC584201) using a BLASTn query. A. japonica was also detected in seeds of the lot used to grow the affected arugula crop. Pathogenicity of a single isolate was tested by inoculating four 37-day-old plants each of arugula, cabbage (Brassica oleracea L. var. capitata), and broccoli (B. oleracea L. var. botrytis L.). Inoculum was obtained from 11-day-old cultures of the isolate grown at 24°C on half-strength APDA. Half of a 2.5 cm diameter agar plug containing hyphae and conidia was ground in 2 ml of sterilized water, and the volume of water increased to 45 ml. Leaves of four plants/host species were sprayed with 3.5 to 4.0 ml of inoculum. The inoculated plants and four control plants of each species treated similarly with sterilized water were immediately incubated in a dark dew chamber at 23°C. After 72 h in the dew chamber, inoculated plants of all three hosts produced similar symptoms of wilting, water soaking, and dark green leaf spotting as the original symptomatic field plants. Conidia formed in the leaf spots on both sides of inoculated leaves. A. japonica was re-isolated from all of the inoculated plants but from none of the symptomless control plants using the method previously described. Pathogenicity tests were repeated, with similar results. Although reported in Italy in 2013 (1), to our knowledge, this is the first report of A. japonica on arugula in the United States. References: (1) G. Gilardi et al. Acta Hort. 1005:569, 2013. (2) E. G. Simmons. Page 368 in: Alternaria, An Identification Manual. CBS Fungal Biodiversity Centre, Utrecht, 2007. (3) S. Werres et al. Z. Planzenkr. Pflanzensh. 108:113, 2001. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

scholarly journals First Report of Tomato chlorosis virus in Tomato in Costa Rica

Plant Disease ◽  
2009 ◽  
Vol 93 (9) ◽  
pp. 970-970 ◽  
Author(s):  
R. M. Castro ◽  
E. Hernandez ◽  
F. Mora ◽  
P. Ramirez ◽  
R. W. Hammond
Keyword(s):  
Costa Rica ◽  
Sequence Analysis ◽  
Nucleotide Sequence Analysis ◽  
Rt Pcr ◽  
Greenhouse Whitefly ◽  
Specific Primers ◽  
First Report ◽  
Sequence Identity ◽  
Pcr Products ◽  
Tomato Chlorosis Virus

In early 2007, severe yellowing and chlorosis symptoms were observed in field-grown and greenhouse tomato (Solanum lycopersicum L.) plants in Costa Rica. Symptoms resembled those of the genus Crinivirus (family Closteroviridae), and large populations of whiteflies, including the greenhouse whitefly Trialeurodes vaporariorum (Westwood), were observed in the fields and on symptomatic plants. Total RNA was extracted from silica gel-dried tomato leaf tissue of 47 representative samples (all were from symptomatic plants) using TRI Reagent (Molecular Research Inc., Cincinnati, OH). Reverse transcription (RT)-PCR reactions were performed separately with each of the four primer sets with the Titan One-Tube RT-PCR Kit (Roche Diagnostics Corp., Chicago IL). Specific primers used for the detection of the criniviruses, Tomato chlorosis virus (ToCV) and Tomato infectious chlorosis virus (TICV), were primer pair ToCV-p22-F (5′-ATGGATCTCACTGGTTGCTTGC-3′) and ToCV-p22-R (5′-TTATATATCACTCCCAAAGAAA-3′) specific for the p22 gene of ToCV RNA1 (1), primer pair ToCVCPmF (5′-TCTGGCAGTACCCGTTCGTGA-3′) and ToCVCPmR (5′-TACCGGCAGTCGTCCCATACC-3′) designed to be specific for the ToCV CPm gene of ToCV RNA2 (GenBank Accession No. AY903448) (2), primer pair ToCVHSP70F (5′-GGCGGTACTTTCGACACTTCTT-3′) and ToCVHSP70R (5′-ATTAACGCGCAAAACCATCTG-3′) designed to be specific for the Hsp70 gene of RNA2 of ToCV (GenBank Accession No. EU284744) (1), and primer pair TICV-CP-F and TICV-CP-R specific for the coat protein gene of TICV (1). Amplified DNA fragments (582 bp) were obtained from nine samples, four from the greenhouse and five from the open field, with the ToCV-p22 specific primers and were cloned into the pCRII TOPO cloning vector (Invitrogen, Carlsbad, CA). Nucleotide sequence analysis of all purified RT-PCR products verified their identity as ToCV, sharing 99.5 to 100% sequence identity among themselves and 96% to 98% sequence identity with previously reported ToCV p22 sequences from Florida (Accession No. AY903447), Spain (Accession No. DQ983480), and Greece (Accession No. EU284745). The presence of ToCV in the samples was confirmed by additional amplification and sequence analysis of the CPm (449-bp fragment) and Hsp70 (420-bp fragment) genes of ToCV RNA2 and sharing 98 to 99% sequence homology to Accession Nos. AY903448 and EU284774, respectively. One representative sequence of the p22 gene of the Costa Rican isolate was deposited at GenBank (Accession No. FJ809714). No PCR products were obtained using either the TICV-specific primers nor from healthy tomato tissue. The ToCV-positive samples were collected from a region in the Central Valley around Cartago, Costa Rica. To our knowledge, this is the first report of ToCV in Costa Rica. The economic impact on tomato has not yet been determined. Studies are underway to determine the incidence of ToCV in Costa Rica field-grown and greenhouse tomatoes. References: (1) A. R. A. Kataya et al. Plant Pathol. 57:819, 2008. (2) W. M. Wintermantel et al. Arch. Virol. 150:2287, 2005.

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