Incidence Of Symptomatic Plants Research Articles

First Report of Alternaria alternata Causing Leaf Blight on Elsholtzia ciliata in China.

Elsholtzia ciliata is an annual medicinal plant characterized to the family Lamiaceae Martinov. It is grown in most parts of China and has high economic value as a traditional Chinese medicine. In September of 2022, E. ciliata plants located at the planting base of traditional Chinses medicine in Daying county (30°35'40″N, 105°14 12″E), Sichuan Province, China, were recorded with leaf blight. The incidence of symptomatic plants was 15% (30 infected plants out of 200 surveyed). The symptoms included an irregular necrotic lesion at the tip of the leaf, which gradually expanded across the entire leaf. To elucidate the cause of the symptoms, 12 symptomatic leaves were sampled from four different plants and 5×5 mm section, including symptomatic and non-symptomatic tissue was excised. Tissue samples were disinfected in 75% ethanol for 30s, and 7% sodium hypochlorite for 1 min, and then rinsed three times with sterile distilled water (Sun et al. 2022). The sampled tissues were placed onto potato dextrose agar (PDA) and incubated at 25℃ in the dark. Seven days later, single spores were recovered onto fresh PDA (Zhu et al. 1992). Colonies on PDA initially appeared white, developing grayish-green conidia with white margins. Conidia (n=150) were collected and observed under the microscope. The conidia were smooth walled and dark brown, with pear-shaped, 12.1-31.4 × 5.0-9.4μm, with 3-5 transverse septa, 1-3 longitudinal or oblique septa. Conidiophores were thick, dark brown, simple with multiple conidial scars, 5.0-75.5 × 2.5.0-5.0μm. Based on morphological observations the 12 isolates were most similar to Alternaria alternata (Simmons 2007). The internal transcribed spacer (ITS) rDNA regions, glyceraldehyde-3-phosphate dehydrogenase (gpd), Alternaria major allergen (Alt a 1), RNA polymerase second largest subunit gene (RPB2) and translation elongation factor 1-alpha (TEF 1) were amplified and sequenced using the primers ITS4/ITS5, RPB2-5F/RPB2-7CR, gpd1/gpd2, EF1-728F/EF1-986R, and Alt-for/Alt-rev respectively (Woudenberg et al. 2015). The sequences of representative isolate (XR) were uploaded in GenBank (ITS: OM319521, RPB2: OM849248, gpd: OM296240, TEF1: OM238122, and Alt a 1: OM649814). The bootstrap value of the isolate and the type strain CBS 595.93 (ITS: KP124320, RPB2: KP124788, gpd: KP124175, TEF1: KP125096, and Alt a 1: JQ646399) on the phylogenetic tree was 99%. Therefore, based on morphology and phylogenetic analysis the fungus was identified as A. alternata. To verify pathogenicity, a spore suspension (1 × 106 conidia/ml) of the representative isolate XR was misted onto the foliage of six twenty-day-old non-symptomatic plants. Six additional plants were sprayed with distilled water and used as controls. The plants were covered with plastic bags for 48 h and incubated at a temperature of 28℃ in the dark. Eight days later, all inoculated plants demonstrated similar symptoms as recorded on the original source, while the control plants were symptomless. The experiment was repeated three times with similar results. A. alternata was re-isolated from the artificially inoculated plants, hence fulfilling Koch's postulates. To our best knowledge this is the first report of leaf blight caused by A. alternata in China on E. ciliate. The disease may be an economic threat and should be further monitored and studied.

Alocasia macrorrhiza Represents a New Host of 'Candidatus Phytoplasma asteris'-Related Strains Associated with Yellows Symptoms in China.

Alocasia macrorrhiza, which belongs to the Araceae family, is an important landscape plant in China, and has of significant medicinal uses. In 2022, A. macrorrhiza displaying abnormal symptoms were found in Qionghai, Hainan Island of China (110°23'3.06″，19°7'56.29″). The incidence of symptomatic plants was about 40% in the sampled areas. The abnormal symptoms included that the ovoid leaves color turned yellow from green gradually, with ovoid leaves chlorosis, mesophyll tissue yellowing, miniature leaves and systemic wilting. The diseased symptoms suspected to be associated with phytoplasma according to the protocols of phytoplasma identification. In order to identify the pathogen, eleven diseased samples and three asymptomatic samples were collected from an area of about 40 hectares. Total DNAs were extracted from 0.10 g fresh plant leaf tissues using a CTAB DNA extraction method. PCR amplifications were performed using primers R16mF2/R16mR1 and fTuf1/rTuf1 specific for the phytoplasma 16S rRNA and tuf genes. Target PCR amplicons were obtained from the DNA of 11 diseased samples, whereas not from the DNA of the asymptomatic samples. The PCR products were cloned and sequenced by Biotechnology (Shanghai) Co., Ltd. (Guangzhou, China), and the obtained sequences were assembled, edited and analyzed using the EditSeq program and DNAMAN version 6.0. The phytoplasma 16S rRNA and tuf gene amplicons were 1336 and 930 bp in length, respectively. The sequences of all 16S rRNA and tuf amplicons in this study were identical. The sequencing data were deposited in GenBank with accession numbers OR466206 (16S rDNA) and OR513090 (tuf). According to the methods and protocols of phytoplasma identified and classification, the phytoplasma strain was described as Alocasia macrorrhiza yellows (AmY) phytoplasma, AmY-hn strain. BLAST search were conducted based on 16Sr RNA and tuf genes. The results showed that the AmY-hn had 100 % 16Sr RNA sequence identity (1336 bp out of 1336 bp) with that of 16SrI-B subgroup phytoplasmas like onion yellows phytoplasma (OY-M, AP006628). The AmY-hn had 100 % tuf sequence identity (930 bp out of 930 bp) with that of 16SrI-B subgroup phytoplasmas like OY-M. RFLP profiles obtained with iPhyClassifier demonstrated that AmY-hn strain was a member of the 16SrI-B subgroup with a similarity coefficient 1.00 to the reference phytoplasma strain (AP006628). Separated phylogenetic analysis based on 16S rRNA and tuf genes obtained with MEGA 7.0 using the neighbor-joining (NJ) method with 1000 bootstrap value indicated that AmY-hn clustered into one clade with phytoplasma strains of OY-M and chinaberry witches'-broom (KP662119) with 100 % and 87 % bootstrap value respectively. To our knowledge, this is the first report that a 'Candidatus Phytoplasma asteris'-related strain belonging to 16SrI-B subgroup infects A. macrorrhiza in China. The 16SrI-B subgroup 'Candidatus Phytoplasma asteris'-related strains can spread outwards through the plant A. macrorrhiza. Thus, the findings in the study will be beneficial to the detection of phytoplasmas which parasitic in this plant and the epidemic monitoring of the related diseases.

First Report of Cucurbit Yellow Vine Disease Caused by Serratia marcescens on Cucurbits in New York.

Cucurbits are one of the most significant commodities in New York, with a value of $92.3 million in 2021 (NASS-USDA 2021). In August 2021, several acorn squash (Cucurbita pepo) cultivar Turbinate plants at Cornell AgriTech research farm in Geneva, NY, had chlorotic, wilting leaves, and older leaves appeared scorched. The phloem of stems, bisected at the crown, had a honey-brown discoloration. The incidence of symptomatic plants was 22% in a one-acre planting field. Most of the symptomatic plants rapidly declined and died. The following year, similar symptoms were observed on muskmelon (Cucumis melo), acorn squash, and winter squash (C. pepo) cultivar Bush Delicata at the same location. These symptoms were typical of Cucurbit Yellow Vine Disease (CYVD) caused by the Gram-negative bacterium Serratia marcescens (Bruton et al. 1998, 2003). Moreover, a high incidence of squash bugs (vector of CYVD) was observed. To identify the causal agent, 45 stems from the symptomatic Bush delicata plants were collected. Each stem was cut into small pieces (2 to 3 mm), surface sterilized with 70% ethanol for 60 sec, 10% bleach for 60 sec, and rinsed with sterile water. The tissue was macerated in sterile water, and the resultant suspension was streaked on King's B (KB) medium (King et al. 1954). Plates were incubated at 28°C for 24 h, and 11 developed white, round bacterial colonies that were smooth and creamy in appearance. Single colonies were transferred to new KB plates and incubated for 24 h. The genomic DNA of two isolates (22212 and 22213) was extracted with the Wizard® Genomic DNA Purification Kit Protocol (Promega, Madison, WI). PCR was carried out using YV1 and YV4 primers specific to the 16S rDNA region of S. marcescens and 79F/R primers specific for S. marcescens causing CYVD (Zhang et al. 2005). The DNA sequence of each PCR product was obtained using Sanger sequencing and submitted to GenBank. Accessions OQ584799 and OQ584800 for YV1/YV4 (isolates 22212 and 22213, respectively) exhibited 100% identity to S. marcescens (384/384 bp, nearest accession identity: CP083754). Accession numbers OQ693911 and OQ693912 for 79F/R showed 99% identity to S. marcescens isolates (309/313 bp, nearest accession identity: CP033623). To fulfill Koch's postulates, Bush Delicata squash plants were grown for two weeks in a greenhouse, and three plants per isolate were inoculated using S. marcescens 22212 and 22213, three plants with Escherichia coli DH5a as a non-pathogenic control, distilled water as a mock-inoculated control, and a noninoculated control. Inoculation was performed by taking a single bacterial colony with a small pin and puncturing the plant's lower stem four to five times (Bruton et al. 2003). Twenty-eight days after inoculation, three of the six plants inoculated with the two S. marcescens isolates (two from 22212 and one from 22213) developed CYVD symptoms as observed in the field. Isolations were made from the stems of symptomatic plants and the mock-inoculated controls. PCR was conducted using YV1/YV4 primers and 79F/R primers (Zhang et al. 2005). Only isolations from symptomatic plants amplified with these primers and PCR products were sequenced. These sequences were identical to the original isolates. To our knowledge, this is the first report of CYVD and phytopathogenic S. marcescens in New York. The impact of CYVD can be substantial, with losses up to 100% (Zhang et al. 2005). Therefore, more knowledge on S. marcescens is needed to determine its biology and prevalence in New York.

First Report of Cowpea Polerovirus 2 and Southern Cowpea Mosaic Virus Infecting Cowpea in Nepal.

Cowpea (Vigna unguiculata L. Walp) is one of the important legume crops of Nepal, which is consumed as a green vegetable or a dried pulse. In recent years, virus diseases have caused significant yield and quality losses in cowpea. In September 2019, five cowpea plants showing virus-like symptoms of mosaic, yellow mosaic, vein clearing, chlorotic spots, (Fig. S1) were collected in Chitwan, Nepal. The incidence of symptomatic plants in the three kitchen gardens was about 10-20%. To identify the viruses associated with the disease, a pooled sample from all five plants was screened initially by next generation sequencing (NGS). Total RNA was extracted from the symptomatic leaves using RNeasy Plant Mini Kit (Qiagen, Germany) and a transcriptome library was generated using the TruSeq Stranded Total RNA LT Sample Prep kit (Illumina, San Diego, CA) according to the standard protocol. NGS was performed using an Illumina NovaSeq 6000 system (Macrogen Inc. Korea). A total of 324,807 contigs in the range of 201-14,645 nucleotides (nt) were obtained and analyzed against the viral reference genome database in GenBank by BLASTn and BLASTx search. Among the analyzed contigs, two large contigs showed homologies to cowpea polerovirus 2 (CPPV2) and southern cowpea mosaic virus (SCPMV. The CPPV2 contig (361,121 mapped reads, mean read coverage of 9,206.4 times) had a nearly complete genome sequence of 5,923 nt and showed 96% identity (99% coverage) with CPPV2 isolate BE179 (GenBank Acc. No. KY364847) isolaed from cowpea in Burkina Faso (Palanga et al. 2017). The SCPMV contig (10,612 mapped reads, mean read coverage of 384.1 times) had a nearly complete genome sequence of 4,172 nt and showed 90% identity (100% coverage) with SCPMV isolate C (GenBank Acc. No. M23021) isolated from cowpea in the USA (Wu et al. 1987). Additionally, mungbean yellow mosaic India virus (MYMIV, genus Begomovirus) and bean common mosaic virus (BCMV, genus Potyvirus) were detected at very low read depths. To confirm the presence of these viruses, total RNA was extracted from individual leaf samples, and reverse transcription PCR (RT-PCR) was performed using specific primers for each virus (Table S1). Three of five cowpea samples were positive for CPPV2, and they were co-infected with one other virus; SCPMV, MYMIV, or BCMV (Fig. S1). One cowpea sample was positive for the remaining one with symptom of overall chlorosis was negative for all four viruses. The amplified products of 1,205 bp for CPPV2 isolates, CPPV2-NP10, -NP12, and -NP24 were sequenced and deposited in GenBank under accession nos. MZ318692-93. These Sanger sequences shared 99% nt identity with the NGS-derived sequence. The amplified product of 1,394 bp for SCPMV isolate SCPMV-NP12 (GenBank acc. no. MZ355623) shared 100% nt identity with the NGS-derived sequence. CPPV2 is a member of the genus Polerovirus and it was first identified and characterized by molecular assays in Burkina Faso (Palanga et al. 2016; 2017). SCPMV is a member of the genus Sobemovirus and it has been reported in the USA, China, and Burkina Faso (Hull et al. 2000; Lee et al. 2001; Wu et al. 1987). In Nepal, MYMIV has been reported in legumes, such as kidney bean, black gram, and mungbean, and BCMV in common bean (Acharya and Regmi 2020). To our knowledge, this is the first report of CPPV2 and SCPMV in cowpea in Nepal. Further work is required to determine the distribution, pathological properties, and economic impact of these two viruses.

Natural Infection of Cichorium intybus (Asteraceae) by Groundnut Ringspot Virus (Genus Orthotospovirus) Isolates in Brazil.

Leaf chicory (Cichorium intybus L.) is a nutritionally rich vegetable used in regional cuisine in Brazil. Plants of C. intybus displaying symptoms (viz. chlorotic and necrotic ringspots, mosaic, and leaf deformation) similar to that induced by orthotospoviruses (genus Orthotospovirus, family Tospoviridae) were observed in three fields (≈ 0.2 ha each) in Gama County, in the Federal District, Brazil, from September 2016 to January 2020 in plants of the cultivars 'Folha-Larga' and 'Spadona' (Fig. 1). Incidence of symptomatic plants was nearly 10% in each field. Transmission electron microscopic examination of thin sections from symptomatic leaf samples showed typical membrane-bounded orthotospovirus particles within cisternae of spongy parenchymal cells (Fig 2). Two individual leaf samples per field were collected and submitted to dot enzyme-linked immunosorbent assay with polyclonal antisera against N protein of tomato spotted wilt virus (TSWV), groundnut ringspot virus (GRSV) and tomato chlorotic spot virus (TCSV). Symptomatic samples strongly reacted only against GRSV antibodies. Total RNA was extracted (Trizol®, Sigma) from all six samples and used as template in RT-PCR assays. The primer J13 (5'-CCCGGATCCAGAGCAAT-3') was employed for cDNA synthesis using M-MLV reverse transcriptase. PCR assays were done with the primer pair BR60/BR65 (Eiras et al., 2001) to obtain ≈ 500 bp fragment of untranslated region and partial N gene in the S RNA segment from each sample. Purified RT-PCR products of two randomly selected individual samples were directly sequenced (GenBank MW467981 and MZ126602) and their BLASTn analyses displayed 99 to 100% nucleotide identity to GRSV isolates previously reported infecting C. endivia L. in Brazil (Jorge et al., 2021). Our analyses combining N protein serology and N-gene sequencing (both directed to the S RNA segment) allowed us to confirm the GRSV infection of C. intybus, but the potential reassortant nature of these isolates (Webster et al., 2015; Silva et al., 2019) are unknown since their M RNA segments were not characterized. Individual leaf extracts (in phosphate buffer, pH 7.0) of the sequenced isolates were mechanically inoculated onto ten seedlings of two C. intybus cultivars ('Folha Larga' and 'Pão-de-Açúcar') and three plants each of the indicator hosts Capsicum chinense PI 159236, Nicandra physalodes; Nicotiana rustica; Datura stramonium; and tomato cv. Santa Clara. Systemic chlorotic and necrotic ringspots, mosaic, and leaf deformation developed in the indicator hosts and infection by GRSV was confirmed via serological assays 20 days after inoculation. However, no symptoms and no serological reaction to GRSV antibodies were observed on the C. intybus cultivars even after two successive mechanical inoculations. This transmission failure might be due to factors such as the requirement of the thrips vector(s), physicochemical barriers in the foliage or the presence of non-mechanically transmissible helper agent(s) necessary to ensure GRSV infection of C. intybus. The natural infection of C. intybus by a not fully characterized orthotospovirus (mostly likely TSWV) has been observed since 1938 in Brazil (Kitajima, 2020). Our report of GRSV infecting C. intybus is thus confirming previous speculations that similar symptoms in this vegetable crop were induced by orthotospovirus infection in Brazil.

First Report of Pectobacterium punjabense Causing Potato Soft Rot and Blackleg in Serbia.

Soft rot and blackleg are common diseases affecting potato (Solanum tuberosum) production in Serbia. Pectinolytic plant pathogens belonging to the genera Pectobacterium cause soft rot and wilt diseases by plant cell wall degradation. These opportunistic phytopathogens lead to considerable economic losses in many potato-growing regions worldwide and are listed among top 10 plant pathogenic bacteria (Mansfield et al. 2012). Potato plants (cv. VR808) with symptoms of wilting, slow growth, stem blackening and tubers softening, were collected from a commercial potato field in Zobnatica (Serbia) in July 2019 and subjected to analysis. All symptoms occurred in the same field and the incidence of symptomatic plants was approximately 5%. Isolation was performed from 10 randomly chosen potato plant and tuber samples, expressing wilting and soft rot symptoms. Plant tissue was surface-disinfected and 1 cm length sections from the margins of lesions were macerated in sterile distilled water for 25 min and streaked on nutrient-agar medium. After 48 h of incubation at 26°C, predominant shiny, cream-colored, round colonies were obtained from all samples. Three representative isolates (MMZKVR1, MMZCVR2, and MMZKVR3) from independent samples were selected randomly and subjected to biochemical and pathogenicity tests. Isolates were gram-negative, nonfluorescent facultative anaerobes, exhibiting pectinolytic activity on potato tuber slices and hypersensitive response on tobacco leaves. They expressed catalase activity but did not express oxidase or acid phosphatase activity or produce indole. All strains grew at 37°C, in 5% NaCl, and reduced nitrate. Pathogenicity of the obtained isolates was tested on 3-week-old healthy potato plants (cv. VR808 and cv. Kiebitz) grown in commercial Baltic Tray Substrate (Hawita) in the greenhouse, as well as on potato tubers of the same varieties. Three potato plant stems per isolate were inoculated by the toothpick piercing method (Duarte et al. 2004) using bacterial suspension (approx. 1 × 108 CFU/ml). Inoculated plants were incubated under plastic bags in a greenhouse at 25 ± 2°C. Blackleg symptoms and stem wilting developed 48 hours after inoculation. No symptoms were observed on plants inoculated with sterile toothpicks dipped in sterile distilled water. The pathogen was re-isolated from symptomatic plants, fulfilling Koch's postulates and sequencing of 16S rDNA confirmed the originally isolated pathogen. Three potato tubers per isolate were inoculated by toothpicks dipped in bacterial suspension (approx. 1 × 108 CFU/ml). Inoculated tubers were placed in a sealed plastic container at 25 ± 2°C. Treatment with sterile distilled water was used as a negative control. Softening of the tissue around the inoculation point developed within 48 h from inoculation, and no symptoms developed on the control tubers. For molecular analyses, total DNA of the isolates was extracted using the DNeasy Plant Mini Kit (Qiagen). The isolates were not detected in diagnostic PCR assays using specific primers Br1F/L1R for the detection of P. brasiliense (Duarte et al. 2004) and primers EXPCCF/EXPCCR for P. catotovorum subsp. carotovorum (Kang et al. 2003). The 16S rDNA PCR amplification was performed using the universal PCR primer pair 27F/1492R (Fredriksson et al. 2013) and followed by Sanger sequencing (Macrogen Europe BV). The BLASTn analysis of sequences (GenBank Accession Numbers MZ048661, MZ048662, and MZ157274) revealed 100% query coverage and 100% identity to the sequences of Pectobacterium punjabense in NCBI (MT242589 and CP038498) isolated from potato in China and Pakistan (Sarfraz et al. 2018), respectively. All three obtained isolates were proposed to belong to Pectobacterium punjabense sp. nov. To further validate the identification, isolate MMZCVR2 of P. punjabense was selected for multilocus sequence analyses of 5 housekeeping genes (gyrA, recA, recN, rpoA and rpoS). The gyrA (MZ161817), recA (MZ161818), recN (MZ161819), rpoA (MZ161820) and rpoS (MZ161821) sequence analysis showed the highest nucleotide identity (99.44 to 100%) with P. punjabense strain SS95 (Sarfraz et al. 2018) previously deposited in NCBI GenBank database. To our knowledge, this is the first report of blackleg and soft rot caused by P. punjabense on potato in Serbia. Pectobacterium punjabense is a newly described species causing soft rot and blackleg disease in potato plants (Sarfraz et al. 2018). Its current geographic distribution is not well-described but important to know since soft rot bacteria are easily transported long distances in latently infected seed tubers and can cause significant economic losses in potato production worldwide.

Olive tree represents a new host of a subgroup 16SrVII-B phytoplasma associated with witches' broom disease in Brazil.

Olive trees exhibiting slow development, yellowing, and high intensity of shoot proliferation with small leaves were observed in commercial plantings, in the municipality of Extrema, Minas Gerais (MG) state in 2015. The incidence of symptomatic plants was about 70% and diseased trees presented yield reduction. Here we report the association of symptomatic olive trees with a phytoplasma and describe its molecular identification. Symptomatic plants (38 trees) were sampled in three growing areas located in the same municipality. The samples consisted of bunch of leaves and young shoots. The total DNA was extracted using DNeasy® Plant Mini Kit (Qiagen, Hilden, Germany). Phytoplasma detection was conducted by nested PCR with primers P1/16S-SR (Lee et al. 2004) followed by R16F2n/R16R2 (Gundersen and Lee 1996). PCR assays generated amplicons (~1.2 kb) from 28 trees out of 38 symptomatic plants, confirming the association of phytoplasma with diseased plants. The disease was named olive witches' broom. The genomic fragments amplified by nested PCR were cloned into Escherichia coli DH5α and sequenced. The sequence representative of the olive phytoplasma was designated OWB-Br01 (Olive Wiches' Broom-Brazil 01) and deposited in GenBank under accession number MH141985. This sequence shared 99% sequence identity with phytoplasmas affiliated with 16SrVII group. According to the iPhyClassifier online tool (Zhao et al. 2009) the olive witches'-broom phytoplasma was classified as a variant of subgroup 16SrVII-B with a pattern similarity coefficient of 0.99. The phylogenetic tree showed that OWB-Br01 phytoplasma emerges from the same branch of the reference phytoplasma of the 16SrVII-B subgroup (Erigeron witches᾽-broom phytoplasma - GenBank AY034608), indicating that the olive tree phytoplasma is a member of the 16SrVII-B subgroup. The pathogenicity test was performed with 28 healthy plants (cultivar Arbequina) grown in pots, which were grafted by simple english forklift with scions obtained from olive plants (Arbequina) six years old, naturally infected by the phytoplasma. The initial symptoms were observed four months after grafting and at eight months 22 grafted plants exhibited slow growth, yellowing, and small leaves as those naturally observed in the fields. Molecular characterization allowed identify the phytoplasma as a member of the 16SrVII-B subgroup. In Brazil, representatives of the 16SrVII group were previously reported in association with diverse botanical species. Thus, a strain of 16SrVII-C subgroup was identified in sunn hemp (Flôres et al. 2013); the reference phytoplasma of 16SrVII-D subgroup was found in erigeron plants (Flôres et al. 2015); and the representative of 16SrVII-F was detected in the wild species Vernonia brasiliana. (Fugita et al. 2017). Specifically regarding subgroup 16SrVII-B, the reference phytoplasma of this subgroup was described from erigeron and periwinkle (Barros et al. 2002), while other members of this subgroup were reported in cauliflower (Pereira et al. 2016a) and ming aralia (Pereira et al. 2016b). The disease here studied is a threat since olive planting is in large expansion in Brazil. A potential control option could be use of propagative material from sources free of the pathogen. Based on our findings, olive tree represents a new host for subgroup 16SrVII-B phytoplasma, which is different from 16Sr groups previously reported as associated with olive witches' broom in other countries.

First Report of Target Spot of Soybean caused by Corynespora cassiicola in the Colombian Eastern Plains.

Soybean (Glycine max L. Merr.) is an important leguminous crop for Colombia, given the growing demand from the livestock, poultry, and aquaculture industries. About 80 percent of Colombian soybean production is in the State, or Department, of Meta, located in the Eastern Plains region, or Llanos Orientales, where the crop has an average yield of 2.5 t.ha-1 (FENALCE 2020). In July 2017, foliar symptoms, including rounded to irregular reddish-brown spots surrounded by a yellowish halo progressing to irregular spots with concentric rings, and in severe cases defoliation, were observed in a production field of Soyica P-34 soybean cultivar in Puerto López, Meta (Colombia). Dark brown lesions on stems and dark-brown to black spots on pods were also observed, and the incidence of symptomatic plants was recorded as 50%. Four infected plants were arbitrarily sampled from different locations across the field, and used for pathogen isolation. Specimen collection was conducted according to the permit conferred to AGROSAVIA under ANLAS' resolution No 1466 of December 03, 2014, Colombia. Symptomatic leaf pieces (~ 5 mm2 sizes) were rinsed with tap water for 1 min, dipped in a 0.5% sodium hypochlorite for 2 min and rinsed with sterile distilled water for 1 min, and then plated on potato dextrose agar (PDA, 39 g.L-1). Plates were incubated at 25°C for two weeks with a 12/12 h light/dark cycle using dark light. Four monoconidial isolates, with similar morphological characteristics, were obtained. Observations under the light microscope showed that conidia (n=50) were hyaline, elongated, 65-120 × 9-12 μm, with 3 to 10 pseudosepta, corresponding with those characteristics described for Corynespora cassiicola (Berk. & M. A. Curtis) C. T. Wei (Ellis and Holliday, 1971). Colonies, on PDA medium, were dark gray with abundant aerial mycelia growth. To confirm the morphological identification, extracted DNA from isolates AGSV13 and AGSV15 was used as a template to obtain partial sequences of the internal transcribed spacer (ITS) region of ribosomal DNA using the primer pair ITS 5/ITS 4 (White et al. 1990). Results from an NCBI-BLASTn search revealed that the ITS sequences (GenBank accessions MN298749 and MN298751) were 100% identical to those of C. cassiicola in GenBank (Accessions MN945374, AB873045). A phylogenetic analysis, using the maximum likelihood method, based on ITS sequences from voucher specimens of Corynespora spp. available at GenBank, revealed that the two isolates were placed in the same clade as C. cassiicola. Pathogenicity tests were conducted by spraying a conidial suspension (1 x 104 conidia.ml-1) of C. cassiicola AGSV13, onto young leaves (two to four true leaf stages) of 10 soybean plants (cv. Soyica P-34). Five plants were sprayed with sterile distilled water and served as non-inoculated control. All plants were incubated at high humidity for seven days at 28°C. Fungal inoculated plants showed typical foliar symptoms of brown spots surrounded by a yellowish halo, similar to those observed in the field. Disease symptoms were not observed on plants of the non-inoculated control. Fungal cultures were recovered from symptomatic leaves of all inoculated plants and verified as similar in morphology to the original C. cassiicola isolates, thus fulfilling Koch's postulates. Based on morphology, pathogenicity, and sequence data results, this is the first report of C. cassiicola causing Target spot on soybean in the Eastern Plains of Colombia and expands our knowledge of this disease. Target spot poses a threat to the expanding soybean crops in this country, as the range of yield losses due to this disease, in South America, has been estimated to be 8% to 42%, depending on the cultivar (Edwards Molina et al. 2019). These findings are significant to the soybean industry in Colombia and will be useful to provide better recommendations to growers for disease monitoring and management.

First Report of Southern Bean Mosaic Virus Infecting Common Bean in Zambia

Southern bean mosaic virus (SBMV, genus Sobemovirus) is one of the causative agents of mosaic and mottle diseases in common bean (Phaseolus vulgaris) (Tremaine and Hamilton 1983). The ∼4.2 kb (+)ssRNA genome of SBMV is encapsidated in isometric virions of about 30 nm in diameter (Tremaine and Hamilton 1983). During surveys conducted in the Eastern Province of Zambia in April 2018, several common bean plants were observed with virus-like symptoms such as vein clearing, leaf rolling, mosaic, and leaf narrowing. Incidence of symptomatic plants across 20 fields ranged from 23 to 100%, averaging 67.5%. A total of 121 symptomatic leaf samples were collected from 20 farmers’ fields and preserved dry on silica gel. Sixteen samples were randomly selected and pooled into one sample (Mse-1) for total nucleic acid (TNA) extraction as described (Djami-Tchatchou and Straker 2012). The TNA was preserved in RNAStable (Biomatrica, Japan) and shipped to Inqaba Biotechnical Industries (Pretoria, South Africa) for high-throughput sequencing (HTS). HTS was achieved via cDNA synthesis and library construction with the Illumina TruSeq RNA Library Prep Kit (Illumina, San Diego, CA). The cDNA library was sequenced on the Illumina MiSeq platform, generating 4.1 million single raw reads of ∼300 nucleotides (nt) each. De novo assembly of 188,160 virus-aligned reads and their subsequent use to query GenBank with BLASTn resulted in the detection of SBMV, bean common mosaic necrosis virus, bean common mosaic virus, cowpea aphid-borne mosaic virus, and cucumber mosaic virus in the sample. To confirm SBMV occurrence and also validate the HTS results, a pair of virus-specific primers (SBMV-1F, 5′-AGCTGGATTTCCTACCTTTGTG; SBMV-1R, 5′-GGCGTCATCTCCGTTTATCTT) was designed to amplify a 873-bp fragment encompassing 32 bp of the 5′ untranslated region, a 492-bp movement protein (ORF1), and a 353-bp partial P2a gene (ORF2) of SBMV by reverse transcription PCR. Each of the 121 samples was screened for SBMV along with the other four viruses (Ha et al. 2008; Niimi et al. 2003). Twenty-five of the 121 samples (20.7%) tested positive only for SBMV, whereas the remaining 96 samples had different combinations of the other four viruses (data not shown). The SBMV-positive samples had mosaic and vein clearing symptoms. The purified PCR products from three samples (EP-88, Mse-13, and Mse-17) were directly Sanger sequenced and deposited in GenBank (MN653952 to MN653954). Pairwise distance analysis revealed that the three Sanger-derived sequences shared 81 to 100% nt identities with each other. The 4,100-bp-long near-complete genome of SBMV was independently Sanger sequenced from sample Mse-13 (MN326873) and determined to encode all the four predicted ORFs of the virus. In pairwise comparisons, the Mse-13 sequence shared the highest (97.2%) and lowest (89.3%) nt sequence identities with the resistance-breaking strain SBMV-B(ARK) (AF055887) from Arkansas and isolate TZ (MG344643) from Tanzania, respectively. SBMV has been previously reported in common bean in many countries across several continents (Verhoeven et al. 2003). However, to the best of our knowledge, this is the first report of SBMV infecting common bean in Zambia. The result adds to the repertoire of viruses circulating in farmers’ fields in Zambia and further confirms the complexity of probable viruses contributing to suboptimal common bean yields in the country.

DETECTION OF TURNIP MOSAIC VIRUS INFECTING MACA IN YUNNAN, SOUTHWEST OF CHINA

Maca (Lepidium meyenii), also known as Peruvian gin- seng, is a herbaceous biennial plant of the crucifer family native to the high Andes. During a survey in November 2014, maca plants showing dwarfing and thickening of the main leaf veins, were observed in Huize county of Yunnan province (Southwest China). The incidence of symptomatic plants was approximately 50% and aphids were observed in the fields. Two symptomatic plants were collected and tested for the presence of viruses. Flexuous filamentous particles 700-750 nm in length were observed with the electron microscope in leaf dips suggesting the presence of an aphid-transmitted potyvirus. To investigate this hy- pothesis, total RNA was extracted using TRIzol reagent (Invitrogen, USA) and tested by RT-PCR (Chen et al., 2001). The first strand cDNA synthesis was conducted using Po- tyviridae universal primer M4-T (5’-GTTTTCCCAGT- CACGACTTTTTTTTTTTTTT-3’), and PCR was done with primers M4 (5’-GTTTTCCCAGTCACGAC-3’) and S (5’-GG(A/G/C/T)AA(C/T)AA(C/T)AG(C/T)GG(A/G/ C/T)CA(A/G)CC-3’). An amplicon of approximately 1.7 kb was obtained from both diseased samples, indicating the presence of a potyvirus. The RT-PCR products were cloned into pGEM-T Easy vector (Promega, USA) and sequenced. Sequences of 1,689 bp of two isolates were identical and shared 100% nucleotide sequence identity with an isolate of Turnip mosaic virus (TuMV) from China (CHN12) (Gen- Bank accession No. AY090660). The sequence of this maca isolate (TuMV-maca) was deposited in GenBank as accession number KP637171. To our knowledge, this is the first report of TuMV infecting maca.

Temporal Dynamics of Tomato Severe Rugose Virus and Bemisia tabaci in Tomato Fields in São Paulo, Brazil

AbstractTemporal progress of a begomovirus disease in tomato fields and the abundance of its whitefly vector, Bemisia tabaci biotype B, were evaluated during three consecutive tomato plantings in the municipality of Sumaré, state of São Paulo, Brazil, in 2006 and 2007. The incidence of symptomatic plants and the number of adult whiteflies were weekly monitored on experimental plots randomly chosen in tomato commercial fields. Tomato severe rugose virus (ToSRV) was identified as the causal agent of the disease, and its relationships with other Brazilian begomoviruses was confirmed by partial and complete nucleotide sequencing of the viral genome. The disease temporal progress was analysed by fitting different models to disease incidence. The monomolecular model showed the best fit, which is consistent with a predominant role of primary spread in the epidemiology of ToSRV. A higher number of adult whiteflies were observed at the borders of the plots, also suggesting primary spread of ToSRV from external sources of inoculum, which might be represented by weeds and volunteer tomato‐infected plants. In Brazil, since 2004, there is a legislative measure that mandates, for some regions of processing tomato plantings, a 2‐month crop‐free period during the year. Based on our results, we suggest the extension of this measure to all tomato‐producing regions, including fresh market tomato. We also suggest that growers emphasize the elimination of old plants from harvested fields that can serve as virus reservoirs several weeks prior to new plantings and weeds nearby the fields to limit the primary spread of ToSRV.

First Report of Tomato chlorosis virus Infecting Tomato Crops in Uruguay.

A survey of viral diseases was carried out during 2012 to 2013 in two major tomato (Solanum lycopersicum L.) producing regions in Uruguay (Salto and Canelones). Lower leaves of fruit-bearing plants were observed displaying yellowing and interveinal chlorosis under greenhouse conditions. The symptoms were similar to those associated with magnesium deficiency. However, the chlorosis displayed a tendency to move up affecting medial and apical leaves and prevailed even after supplementary magnesium applications to the soil, indicating potential infection by either Tomato chlorosis virus (ToCV) or Tomato infectious chlorosis virus (TICV) (3). Four leaf samples were collected from two sites in Canelones and 28 samples were collected from distinct commercial fields in Salto. Whiteflies (Bemisia tabaci biotype Q and Trialeurodes vaporariorum) were present in all sampling sites. Total RNA was extracted from symptomatic and healthy (control) plants and used for cDNA preparation with the HS-11/HS-12 primer pair followed by PCR amplification using the same primer pair. The 587-bp amplicon, corresponding to a highly conserved region of the heat shock protein (HSP-70) homolog gene reported in both TICV and ToCV genomes, was observed only in the symptomatic samples. These PCR products were then subjected to nested PCR using the ToCV specific primer pair (ToC-5/ToC-6) and TICV specific primer pair (TIC-3/TIC-4) (1). The expected 463-bp ToCV-specific amplicon was observed in all symptomatic plants but not in the healthy controls. The 223-bp amplicon corresponding to TICV was not observed in any sample, indicating the sole presence of ToCV. The amplicon of one Uruguayan ToCV isolate from Salto (named as CRS03) was purified and directly sequenced (GenBank KC626018). BLAST analysis revealed 99% identity of CRS03 with one Spanish isolate (AF233435.1) (2). Virus-free B. tabaci biotype Q adults were exposed to symptomatic plants infected with the CRS03 isolate for a 24-h period and then cage-confined with 10 healthy tomato plants (line 'LT17') for a 48-h period. Symptoms were reproduced in all tested plants after a 65-day period and ToCV infection was confirmed via PCR assays and by sequence analysis of the gel-purified amplicons. This is the first formal report of ToCV infecting tomatoes in Uruguay. Incidence of symptomatic plants in tomato crops varied from 30 to 100%, even under low whitefly pressure. Epidemiological information needs to be generated in order to evaluate the impact of ToCV in the fresh-market tomato yield and quality.

Damping-Off and Root Rot of Pepper Caused by Fusarium oxysporum in Almería Province, Spain.

Bell pepper (Capsicum annuum L.) is intensively cropped in ~9,920 ha of plastic houses in southern Spain. In summer 2013, pepper seedlings cv. Melchor, Acorde, Galena, Prometeo, and Souleria, with 4 to 8 leaves, grown in a nursery greenhouse near El Ejido, Almería Province, exhibited root rot and stunting. Incidence of symptomatic plants was ~35% among over 10 million. Fusarium sp. was consistently isolated on potato dextrose agar (PDA) from primary and secondary roots of symptomatic plants. Eight single spore isolates (FC1, FC2, FC3, FC4, FC5, FC6, FC11, and FC12) were identified on PDA, carnation leaf-piece agar medium, and Spezieller Nährstoffarmer agar medium as Fusarium oxysporum because of their production of macroconidia (20.5 to 38.2 × 3.9 to 5.8 μm) containing mostly three or rarely four septa, with foot-shaped basal cells. Microconidia (5.9 to 16.5 × 2.6 to 4.7 μm) with 0 to 1 septa formed on false heads on short monophialides and chlamydospores. DNA was extracted from various isolates used in the pathogenicity test, and a portion of the elongation translation factor 1-alpha using primers EF-1 and EF-2 was amplified and sequenced. All the pathogenic isolates were identical, and they differed from the non-pathogenic in 6 to 8 base pairs. The isolates had 99% homology with several isolates of F. oxysporum corresponding to different specialized forms (vasinfectum, lilii, lycopersici, and radicis-lycopersici) at the Fusarium-ID database (1) and GenBank. The sequences of two isolates, FC-6 and FC-12, were deposited in GenBank with accession nos. KF928930 and KF928931, respectively. The pathogenicity of these eight isolates was tested on pepper cv. Melchor in 1-liter containers filled with vermiculite in August and October. Seedlings were inoculated at sowing. PDA plates fully covered with the colony of each isolate were separately blended and homogenized with 300 ml of sterile distilled water. Inocula (5.0 × 105 to 9.4 × 106 conidia/ml) were poured at 50 ml per container. Each experiment had four replicates and 5 to 6 plants per replicate. Treatments with different isolates were arranged in a randomized complete block design. In both experiments, the same number of uninoculated seedlings served as controls. The plants were maintained for 40 days following inoculation in a greenhouse with mean temperatures of 24.0 to 32.4°C and 23.6 to 31.20°C for August and October experiments, respectively. In both experiments, all control plants and those inoculated with FC2, FC3, and FC4 remained asymptomatic. The first wilting occurred 11 days after inoculation. At the end of the August experiment, plants inoculated respectively with FC1, FC5, FC6, FC11, and FC12 showed symptoms in 60, 70, 65, 80, and 90% and 25, 0, 15, 40, and 25% died. At the October experiment, plants showed symptoms in 91.7, 95.8, 100.0, 91.7, and 87.5% and 83.3, 75, 62.5, 83.3, and 79.2% died. Symptomatic plants exhibited damping-off, necrosis of the primary and secondary roots, and sometimes necrotic streaks on the stem. F. oxysporum was consistently recovered from the primary root of symptomatic plants in both experiments and 10 of these isolates were inoculated in a third pathogenicity test, being all pathogens, thus fulfilling Koch's postulates. Although F. oxysporum was reported in peppers (2), to our knowledge, this is the first report of F. oxysporum as the causal agent of damping-off and root rot in pepper seedlings in Almería Province.

First Report of Leek yellow stripe virus in Allium sativum in Western India.

Garlic (Allium sativum L.) is an important bulbous spice crop in India as well as other parts of world. Garlic is well known for its medicinal properties. Degeneration due to viral infections is one of the important constraints in exploiting its yield potential. Leek yellow stripe virus (LYSV), genus Potyvirus, family Potyviridae, is a prominent virus known to infect garlic worldwide (4). During July 2013, potyvirus-like symptoms such as mosaic, streaking, stunting, mottling of leaves were observed on garlic cv. G-41 and landrace Ranibennur local, collected from Karnataka, India, and maintained at the Directorate of Onion and Garlic Research, Rajgurunagar, Pune, India. The incidence of symptomatic plants was estimated at 70% for Ranibennur local and 68% for cv. G-41. The symptomatic leaves were sampled diagonally from the field. Twenty symptomatic plants per cultivar with each sample was composited from young, middle, and lower (basal) leaves of the plant. These samples were tested by double-antibody sandwich (DAS)-ELISA for LYSV using commercially available kit (Agdia Inc., Elkhart, IN). ELISA-positive plants were further subjected to molecular studies. Total RNA from the infected leaf samples were extracted by RNeasy Plant Mini kit (Qiagen GmbH, Hilden, Germany) and assayed by reverse transcription (RT)-PCR using primer pair LYSV-F 2 (5'-GCACCATACAGTGAATTGAG-3') (1), LYSV-R (5'-GCCTCGCGCGCTCTAA-3') (3) to amplify 874 bases of partial Nib and partial coat protein gene. The amplified product of 874 bp derived from A. sativum isolate was purified (QIAquick PCR Purification Kit, Qiagen) and cloned using vector pDrive (Qiagen). The recombinant clones were sequenced and submitted in NCBI database (GenBank Accession No. KF850539). The sequence analysis performed on CLC Main Workbench Version 6.8.4 gave confirmation of LYSV. Further, phylogenetic analysis of the 874-nt sequence revealed the highest nucleotide identity (80 to 82%) with LYSV isolates (DQ925453, JN127339, AB005611, and JX429965). To the best of our knowledge, this is the first report of natural infection of garlic by LYSV in western India. LYSV is known to cause direct losses in garlic and other related Allium spp. Up to 54% reduction in bulb weight was observed due to single infection of this virus (2). Hence, our first report about this virus has significant impact on garlic production scenario, if this virus found to be widespread in the country. For this, additional surveys and genotype screenings are needed to obtain a better understanding of the potential impact of LYSV on garlic production in India.

First Report of 'Candidatus Liberibacter solanacearum' in Carrot in Mainland Spain.

In the summer of 2008, symptoms of leaf curling with yellow, bronze, and purple discoloration, twisting of petioles, stunting of shoots and tap roots, and proliferation of secondary roots were observed in 18 commercial carrot (Daucus carota L.) fields (~62 ha) severely infested with psyllids (mainly Bactericera sp.) from 52 fields (297 ha) located in Alicante and Albacete provinces of Spain. Incidence of symptomatic plants was variable among fields. Similar symptoms were observed in 2009, 2010, and 2011. Symptoms resembled those associated with phytoplasma, spiroplasma, or the bacterium 'Candidatus Liberibacter solanacearum' infections in carrot (1-4). Aster yellows and stolbur phytoplasmas and Spiroplasma citri have previously been reported from carrot in mainland Spain but liberibacter infection has not been documented in this region (1). Studies were conducted to determine if 'Ca. L. solanacearum' was associated with the symptoms. Petiole samples of symptomatic carrot plants were collected in 2009 (25 from 9 fields in Alicante and Albacete provinces) and early 2010 (21 from 8 fields in Alicante, Albacete, and Valencia provinces) from symptomatic fields where incidence ranged from 50 to 90%. In addition, one sample collected in 2008 in Alicante was included in the assay. Also, samples were collected from five asymptomatic carrot plants. Total DNA was extracted from 0.5 g of petiole tissue of each sample with the CTAB extraction buffer method (3,4). DNA extractions were analyzed by PCR assay using primer pairs OA2/OI2c and CL514F/R to amplify a portion of 16S rDNA and rplJ/rplL ribosomal protein genes, respectively, of 'Ca. L. solanacearum' (3,4). DNA samples were also tested for phytoplasmas and S. citri by nested-PCR assays using primer pairs P1/P7 followed by R16F2n/R16R2n and ScR16F1/ScR16R1 followed by ScR16F1A/ScR16R2, respectively (2). A 1,168-bp fragment of 16S rDNA was detected in DNA extracted from 1, 12, and 12 symptomatic samples collected in 2008, 2009, and 2010, respectively, suggesting the presence of 'Ca. L. solanacearum' in the carrot samples. A 669-bp rplJ/rplL fragment also was amplified from DNA of the same samples. Liberibacter was not detected in asymptomatic plants. Eight and two samples were infected with S. citri and aster yellows phytoplasmas, respectively. Three samples were infected with S. citri and 'Ca. L. solanacearum' and one sample was infected with all three pathogens. Three amplicons obtained from the PCR assays with both primer pairs from carrot samples collected in 2009 and 2010 were sequenced directly. BLAST analysis of the 16S rDNA sequences (GenBank Nos. HQ454302, HQ454303, and HQ454304) showed 99% nucleotide identity to those of 'Ca. L. solanacearum' amplified from carrot in Finland (GU373049). The rplJ/rplL nucleotide sequences (HQ454305, HQ454306, and HQ454307) were 97% identical to sequences of the analogous rplJ/rplL 'Ca. L. solanacearum' ribosomal protein gene from carrot in Finland (GU373051). To our knowledge, this is the first report of 'Ca. L. solanacearum' in carrot in mainland Spain and also the first evidence of mixed infections of S. citri, 'Ca. L. solanacearum', and phytoplasmas in carrot.

Ozone-Induced Leaf Symptoms on Vegetation in the Mingo National Wildlife Refuge, Missouri

Abstract Field surveys were conducted during 1998, 2000, 2003, and 2004 within the Mingo National Wildlife Refuge in southeastern Missouri to determine if ambient ground-level ozone was impacting ozone-sensitive refuge vegetation. Ozone-induced leaf symptoms (stipple) were observed within the refuge during each survey year. Percentage of bioindicator plants exhibiting stipple were wild grape (16.1%) > Common Milkweed (16.0%) > ash (7.5%) > Black Cherry (6.7%) > Flowering Dogwood (4.9%) > Sassafras (2.3%) > Sweetgum (1.2%). By year, the incidence of symptomatic plants were 1998 (22.8%) > 2003 (3.9%) > 2000 (3.4%) > 2004 (2.5%). Cumulative ambient ozone levels (SUM60, ppb.hrs) monitored at the closest EPA monitor (Bonne Terre, MO) at time of survey were 1998 (44,886) > 2000 (39,611) > 2003 (38,465) > 2004 (15,147). The cumulative SUM60 threshold value of ozone needed to cause foliar symptoms on ozone-sensitive plants within the refuge appears to be ca. 10,000 ppb.hrs. Ozone injury is likely to occur on ozon...

First Report of Soybean Witches'-Broom Disease Caused by Group 16SrII Phytoplasma in Soybean in Malawi and Mozambique.

Soybean (Glycine max L.) is an important grain legume cultivated on approximately 1.24 million ha in Africa (1). Malawi ranks fourth in area of production in Africa, with 75,000 ha in 2009 (1). Soybean is also gaining importance in Mozambique and several other southern African countries due to diversification programs. During a field survey conducted in March 2010, soybean plants with phyllody and witches'-broom disorders typical of phytoplasma infection were observed in three of five fields surveyed in Lilongwe (Chitedze Research Station) and Salima (Channa, Chitala) districts in Malawi and three of four fields surveyed in Zambezia Province in Mozambique. Symptoms consisted of shoot proliferation, reduced leaflets, shortened internodes, proliferated auxiliary shoots producing witches'-brooms, virescence, and phyllody. Incidence of symptomatic plants was <1% in Malawi and 10 to 15% in Mozambique. Yield loss was 100% in affected plants. Five leaf samples each from symptomatic and asymptomatic plants were collected from six fields; total genomic DNAs were isolated and used as templates in PCR using phytoplasma-universal primer pair P1 and P7 for 16S-23S ribosomal RNA encoding region (3). PCR amplicons (1,709 bp) were produced from only templates derived from symptomatic plants. Amplicons from a symptomatic plant each from Malawi (Channa, Salima District) and Mozambique (Mutequelse, Zambezia Province) were directly sequenced in both directions and submitted to the GenBank (Accession Nos. HQ840717 and HQ845208). Nucleotide sequences of the two African soybean witches'-broom (SoyWB) phytoplasma strains were 100% identical. The virtual restriction fragment length polymorphism (RFLP) pattern derived from these sequences using iPhyClassifier software (4) was similar to the reference pattern of the 16Sr group II, subgroup C (cactus phytoplasma, Accession No. AJ293216), with a pattern similarity coefficient of 0.99. A BLASTn search revealed that the African SoyWB phytoplasma sequences had a nucleotide sequence identity of 99% with those of soybean phytoplasma from Thailand (Accession No. EF193353), cactus phytoplasma from China (Accession No. EU099561), and several other members of 16SrII group. Phylogenetic analysis revealed the clustering of these strains with members of 16SrII group. In 1984, the occurrence of phyllody and witches'-broom symptoms in soybean in Mozambique was reported (2), however, no comprehensive details on the pathogen are available. To our knowledge, this is the first report of phyllody and witches'-broom disease in soybean in Malawi and the first molecular evidence of association of a 16SrII-C group 'Candidatus phytoplasma' with the disease in Malawi and Mozambique. Phyllody and witches'-broom is a destructive disease, and its widespread occurrence can adversely affect soybean production in sub-Saharan Africa. Identification of alternative hosts and vector species would improve our understanding of the disease's epidemiology and contribute to development of appropriate tactics to prevent escalation of this problem into a major disease.

First Report of Iris yellow spot virus on Garlic in India.

Garlic (Allium sativum) is a spice crop of prime importance in India as well as other parts of the world. Iris yellow spot virus (IYSV; genus Tospovirus, family Bunyaviridae) is an important pathogen of onion bulb and seed crops in many parts of the world (3). The virus is also known to infect garlic and other Allium spp. (2-4). IYSV infection of garlic was reported from Reunion Island (4) and the United States (1). In February 2010, straw-colored, spindle-shaped spots with poorly defined ends were observed on the leaves of a garlic crop at the research farm of the Directorate of Onion and Garlic Research in the Pune District of Maharashtra State, India, 105 days after planting. The spots coalesced to form larger patches on the leaves, suggesting possible IYSV infection. Symptoms were visible on older leaves and more prevalent on cv. G-41, G-282, AC50, AC200, AC283, and Godavari than on other cultivars. The incidence of symptomatic plants was estimated at 5% for G-41 and AC-200, 8% for G-282 and AC283, and 10% for AC50. Leaves were sampled from 40 symptomatic plants per cultivar with each sample composited from young, middle, and older (basal) leaves of the plant. Samples were assayed by double-antibody sandwich-ELISA (Loewe Biochemica GmbH, Sauerlach, Germany) and each tested positive for the virus. Total RNA was extracted from the leaves of ELISA-positive plants using the RNAeasy Plant Mini kit (Qiagen GmbH, Hilden, Germany) and tested by reverse transcription-PCR assay using primers IYSV-F (5'-TCAGAAATCGAGAAACTT-3') and IYSV-R (5'-TAATTATATCTATCTTTCTTGG-3') (2) designed to amplify 797 bp of the nucleocapsid (N) gene of IYSV. Amplicons of expected size were obtained and cloned into a pDrive vector (Qiagen GmbH). The recombinant clone was sequenced (GenBank Accession No. HM173691). Sequence comparisons showed 98 to 100% nt identity with other IYSV N gene sequences in GenBank (Nos. EU310294 and EU310286). A phylogenetic analysis of the deduced amino acid sequences of the N gene showed that the garlic isolate of IYSV grouped most closely with onion IYSV isolates from India (GenBank Nos. EU310294, EU310286, EU310300, and EU310296). To our knowledge, this is the first report of natural infection of garlic by IYSV in India. Additional surveys and evaluations are needed to obtain a better understanding of the potential impact of IYSV on garlic production in India.

First Report of Phoma terrestris Causing Red Root Rot on Sweet Corn (Zea mays) in North Carolina.

Red root rot, caused by Phoma terrestris E. M. Hansen, caused premature senescence and yield reductions to fresh-market sweet corn in Hyde County, North Carolina in July 2006. Foliar symptoms developed over a period of 5 to 8 days approximately 1 to 2 weeks after anthesis and included desiccation of leaves and poor development of ears. By 3 weeks after pollination, when the sweet corn was harvested, crowns and the first aboveground internode of affected plants were rotted and reddish colored, but roots appeared normal. The root mass of affected plants tended to be greater than that of unaffected plants. Incidence of symptomatic plants was greater than 30% in some fields and was lower on crops planted and harvested early. Symptomatic and asymptomatic plants were adjacent in affected fields. Diseased plants were more common in fields of sweet corn that followed soybean (Glycine max) or a double-crop of onions (Allium cepa) than in fields that followed corn. Incidence of symptomatic plants also differed among adjacent plantings of different sweet corn hybrids. Hybrids '173A', '182A', '378a', and 'XTH1178' had a high incidence of symptomatic plants and '372A', '278A', '8101', and '8102' were less affected. Samples of symptomatic plants of the hybrid '182A' were examined at the North Carolina Plant Disease and Insect Clinic during August. Olivaceous black pycnidia with long setae around the ostioles were imbedded in the stalk near the first node aboveground. Numerous conidia (1.8 to 2.3 × 4.5 to 5.5 μm) were released in cirri from pycnidia. When cultured on potato dextrose agar (PDA), the fungus produced a red pigment and intercalary and terminal chlamydospores. Pathogenicity was demonstrated in the greenhouse by transplanting corn seedlings or direct-seeding corn into pots of soil infested with plates of PDA containing chlamydospores and hyphae. A suspension of chlamydospores and hyphae also was injected into the stems of plants 28 days after transplanting. Five replicates of the pathogenicity experiments were repeated twice with noninoculated controls. After 8 weeks, P. terrestris was recovered from the roots of all inoculated plants. Soil inoculation resulted in necrotic root tissue in approximately 25% of inoculated plants. Approximately 90% of inoculated plants had discolored crowns that resembled symptoms from field infected plants. Stem inoculations resulted in necrosis extending 2 to 5 cm from the point of injection and resulted in shoot death of 40% of inoculated plants that resulted in the development of an adventitious shoot. Red root rot was prevalent on field corn in the Delmarva Peninsula throughout the late 1980s and 1990s (1). To our knowledge, this is the first report of this disease causing damage to sweet corn in North Carolina. Foliar symptoms and discoloration of crowns of diseased sweet corn plants were similar to previously described symptoms of red root rot on field corn (2), however, roots of affected sweet corn plants were not substantially rotted and did not have a symptomatic reddish pink or dark carmine color, presumably because sweet corn is harvested prior to the development of root symptoms.

Progress, spread and natural transmission of Bahia bark scaling of citrus in Brazil

AbstractProgress, spread and natural transmission of Bahia bark scaling of citrus were evaluated in a trial where 240 screenhouse‐nursed nucellar grapefruit plants –‘Clason’, ‘Little River Seedless’, ‘Red Blush’, ‘Reed’ and ‘Howell Seedless’ cvs – were planted alongside and 5 m apart from a 10‐year‐old symptomatic ‘Marsh Seedless’ grapefruit orchard. Plants were distributed in 16 rows of 15 trees, with three plants of each cultivar per row. Eight trial plants were kept in screen cages. Incidence of symptomatic plants was assessed at 3‐months intervals, for 5 years, and for further 2 years at irregular intervals. Cumulative maps of disease incidence were produced for each assessment date and used in all analyses. Temporal progress was analysed by nonlinear fitting of three disease progress models. Spread was characterised in three levels of spatial hierarchy by the following analyses: ordinary runs, binomial dispersion index, binary power law fitting, isopath mapping and nonlinear fitting of disease gradient models. The first symptomatic plant was detected 2 years after planting. In the last disease assessment, 5 years after the first, 98% of the unprotected plants were symptomatic. None of the screen‐caged trees showed any symptoms. Bahia bark scaling progress was polyetic and best described by the logistic model. Ordinary runs analysis showed little if any evidence of transmission between adjacent trees. Diseased plants showed a very aggregated pattern inside quadrats (D &gt; 5 and b &gt; 1.53). Isopath mapping showed that main spread was only because of the primary inoculum source. Secondary foci were also observed, but they were never dissociated from main initial disease focus. Disease gradient followed wind direction, starting near the original inoculum source and was best described by exponential model. These results support a hypothesis of Bahia bark scaling transmission by air‐borne vectors with limited dispersion ability.