First report of Curvularia leaf spot of field corn, caused by Curvularia lunata, in Mississippi

In July 2021, foliar symptoms characterized by small, circular, light brown to tan lesions (0.5 to 3 mm diameter) with reddish-brown margins were observed on field corn (Zea mays L.) in two commercial fields in Hinds and Marion counties, Mississippi. Disease severity ranged from 2 to 15% on observed leaves. Symptomatic leaves were sealed in plastic bags, stored on ice, and transferred to the laboratory. Lesions were cut into small sections (≈4 mm2) and surface-sterilized with 70% ethanol for 30 s then rinsed with sterile water. Sterilized sections were transferred to potato dextrose agar (PDA) amended with chloramphenicol (75 mg/liter) and streptomycin sulfate (125 mg/liter) and incubated at 25°C in the dark for 7 days. Gray to brown-black colonies with orange margins and melanized, curved conidia with three transverse septa were observed microscopically (Fig. 1; ×400). Conidia measurements ranged from 15 to 25 μm in length and 7.5 to 12.5 μm in width (x̄= 20 × 9.8 μm; n= 44). Colony and conidia morphology were consistent with previous descriptions of Curvularia lunata (Wakker) Boedijn (Mabadeje 1969; Ellis 1971). Pure cultures were obtained, and DNA was extracted from 9-day old cultures. Two isolates (TW003-21; TW008-21) were selected for sequencing of the internal transcribed spacer (ITS) region using ITS4 and ITS5 primers. The 530-bp consensus sequences were deposited in GenBank under the accession No. OK095277 and OK095278. BLASTn queries of NCBI GenBank showed that the sequences shared 100% identity with C. lunata isolate DMCC2087 from Louisiana (MG971304) and isolate CX-3 from China (KR633084). A pathogenicity test was performed on V4/V5 stage corn plants (Progeny 9114VT2P) grown in 10.2 cm pots in the greenhouse. Plants were transferred to a growth chamber one-week prior to inoculation. The two isolates were grown on amended PDA for 14 days at 25°C and an inoculum suspension was prepared for each isolate by rinsing culture plates with 2 ml of autoclaved reverse osmosis (RO) water amended with Tween 20 (0.01%) and re-suspended into 40 ml of RO water containing Tween 20. The final concentration was adjusted to 2.6×105 conidia/ml (TW003-21) and 2×105 conidia/ml (TW008-21). Ten corn plants were sprayed with 10 ml of inoculum suspension for each isolate using a Preval sprayer with a CO2 canister, and 10 plants were sprayed with water containing Tween 20 only. Plants were incubated in a growth chamber at ≈79% relative humidity and 25°C. Foliar symptoms including small, circular, and tan lesions, similar to those observed in the field, developed 3 days after inoculation. No symptoms were observed on control plants. Following incubation, symptomatic leaves were collected and C. lunata was re-isolated as described above. Colony, spore morphology and DNA sequences from inoculated plants were consistent with the original isolates as described above. The disease has been recently reported in Louisiana (Garcia-Aroca et al. 2018), Kentucky (Anderson et al. 2019), and Delaware (Henrickson et al. 2021). Although Curvularia leaf spot has been observed sporadically in MS corn fields since 2009 (Allen, personal communication), to our knowledge, this is the first official report of the disease in MS. While this disease has been more frequently encountered in MS, the economic impact associated with C. lunata is currently unknown. References Anderson, N. R., et al. 2019. Plant Dis. 103:2692. Chang, J., et al. 2020. J. Integr. Agr. 19:551-560. Ellis, M. B. 1971. Dematiaceous Hyphomycetes. Commonwealth Mycological Institute, Kew, England, p. 452-458. Garcia-Aroca T., et al. 2018. Plant Health Prog. 19:140. Henrickson M., et al. 2021. Plant Dis. First Look. Mabadeje, S. A. 1969. Trans. Br. Mycol. Soc. 52:267-271. † Indicates the corresponding author. E-mail: [email protected]

Trunk Surgery as a Tool to Reduce Foliar Symptoms in Diseases of the Esca Complex and Its Influence on Vine Wood Microbiota

In the last few years, trunk surgery has gained increasing attention as a method to reduce foliar symptoms typical of some of the Esca complex diseases. The technique relies on the mechanical removal of decayed wood by a chainsaw. A study on a 14-year-old Cabernet Sauvignon vineyard was carried out to validate the efficacy of trunk surgery and explore possible explanations behind it. Three levels of treatment were applied to three of the most characteristic symptoms associated with some diseases of the Esca complex, such as leaf stripe symptoms (LS), wilted shoots (WS) and apoplexy (APP). The most promising results were obtained by complete trunk surgery, where the larger decay removal allowed lower symptom re-expression. According to the wood types analyzed (decay, medium and sound wood), different changes in microbiota were observed. Alpha-diversity generally decreased for bacteria and increased for fungi. More specifically, main changes were observed for Fomitiporia mediterranea abundance that decreased considerably after trunk surgery. A possible explanation for LS symptom reduction after trunk surgery could be the microbiota shifting caused by the technique itself affecting a microbic-shared biochemical pathway involved in symptom expression.

First report of cucurbit yellow stunting disorder virus and cucurbit chlorotic yellows virus in cucurbit crops in Alabama

During the summer and fall of 2020, foliar yellowing symptoms, including leaf mottle and interveinal yellowing with green veins were observed on several melon, squash, and cucumber plants in commercial fields in Alabama, USA. These foliar symptoms were similar to those caused by the whitefly-transmitted yellowing viruses, cucurbit chlorotic yellows virus (CCYV) and cucurbit yellow stunting disorder virus (CYSDV) (both genus Crinivirus, Closteroviridae). A total of 231 leaf samples showing yellowing, interveinal chlorosis, and mottling (e-Xtra 1, 2) were collected from individual plants from 25 commercial fields in Alabama (70 watermelon, 52 melon, 34 pumpkin, 50 squash, and 25 cucumber) during two sampling periods, June (spring/summer season) and October (fall season) 2020. Total RNA, extracted as described in Tamang et al. (2021), was used in reverse transcription polymerase chain reaction (RT-PCR) with primer sets designed to amplify portions of the CCYV and CYSDV RNA-dependent RNA polymerase (RdRp) genes encoded on RNA1 of these viruses (Mondal et al. 2021, submitted; Kavalappara et al., 2021). Single infections of either CYSDV or CCYV were found in 53 of 57 infected cucurbit samples (of 231 total plants), whereas both viruses were detected in four samples, all squash. In June 2020 near the end of the spring season, CYSDV was identified from 20 of 114 total cucurbit plants tested (17.5%), but CCYV was not identified from any plants. During the fall season, 37 of 117 plants (32%) tested positive for the presence of one or both criniviruses. Of the 37 virus-positive samples from the fall season, 26 were singly infected with CCYV (70%), seven were singly infected with CYSDV (19%), and four were infected with both CYSDV and CCYV (11%). The RdRp amplicon was sequenced from three CCYV-infected plants (2 squash; GenBank Accession No. MZ073347, MZ073348; 1 cucumber, MZ073349) and one CYSDV-infected plant (melon, MZ073350); the 857 nt sequenced portion of the CCYV RdRp gene was found to share 99% identity with the same sequence of CCYV RNA1 isolates from Israel (MH477611.1) and California (MW680157), whereas the 494 nt CYSDV amplicon shared 100% sequence identity with the comparable sequence from RNA1 of a CYSDV isolate from Arizona (EF547827.1). In addition, all of the CYSDV and CCYV infections were confirmed using a second set of primers that amplified 394 and 372 nt sections of the coat protein gene of each virus, respectively (Wintermantel et al., 2009; 2019), encoded on RNA2 of each viral genome. Furthermore, a recently developed multiplex RT-qPCR method (Mondal et al. 2021, submitted) was used to confirm four representative CYSDV and CCYV infections each. This is the first report of CYSDV and CCYV in cucurbit crops from Alabama. Surprisingly, CYSDV was only found in melon plants (20 of 52, 38%), whereas CCYV was only found in squash, pumpkin, and cucumber (26 of 109, 24%); no watermelon plants were infected with either virus, even though watermelon is a known host of both viruses. The identification of CCYV and CYSDV in Alabama, along with a recent report of both criniviruses from nearby Georgia (Kavalappara et al., 2021) illustrates the need for a more thorough sampling of cucurbit crops, further monitoring of the whitefly vector, Bemisia tabaci, and the identification of alternate hosts of these viruses to better understand the epidemiology of these viruses in Alabama and throughout the Gulf Coast region.

Impacts of Sodium Arsenite on Wood Microbiota of Esca-Diseased Grapevines

Although sodium arsenite was widely used in Europe until its ban in 2003, its effects on microorganisms is not clearly understood. To improve our understanding of sodium arsenite curative effect on GTDs, grapevines displaying esca-foliar symptoms from different French regions (Alsace, Champagne, Languedoc) were treated or not with sodium arsenite, and analyzed for their wood microbiota. Using metabarcoding, we identified the fungal and bacterial taxa composition of microbiota colonizing woody trunk tissues. Large differences in fungal microbiota composition between treated and untreated grapevines were observed while no major impacts were observed on bacteria microbiota. The main fungal species detected in untreated necrotic woody tissues was Fomitiporia mediterranea (63–94%), a fungal pathogen associated with esca. The relative abundance of this fungal species significantly decreased after sodium arsenite treatment in the three vineyards, in particular in white-rot necrotic tissues and their borders (−90%). F. mediterranea was the most sensitive to sodium arsenite among fungi from grapevine woody tissues. These results strongly suggest that the effect of sodium arsenite on GTDs is due to its ability to efficiently and almost specifically eliminate F. mediterranea from white-rot necrotic tissues, allowing saprobic fungi to colonize the tissues previously occupied by this pathogenic fungus.

Fomitiporia punicata and Phaeoacremonium minimum associated with Esca complex of grapevine in China

The Esca disease complex includes some of the most important trunk diseases of grapevines (Vitis spp.) and causes serious yield losses in grape production worldwide. However, there has been no detailed study on its presence and associated pathogens in China. During 2017–2019, a preliminary field survey was conducted in eight vineyards in Hebei and Ningxia provinces, China when unusual foliar symptoms were observed. Symptoms were distinct tiger striped leaves, which are typical of grapevine leaf stripe disease (GLSD), one of the most common diseases in the Esca complex. Tiger-stripe leaf symptoms were found in four vineyards, and incidence was cultivar dependent varying with vineyard and year. A total of 266 fungal isolates were obtained from wood tissues of grapevines with typical foliar symptoms of GLSD. Based on morphological characters and multigene-combined phylogenetic analyses, the Ascomycete Phaeoacremonium minimum, one of the pathogens associated with Esca complex was identified. The basidiomycete Fomitiporia punicata, which has never been reported infecting grapevine, was also identified and found to be associated with wood rot in grapevine. The remaining isolates included some known wood pathogens, such as Neofusicoccum species and Diaporthe species. Koch’s postulates were performed in the greenhouse, confirming that both F. punicata and P. minimum caused leaf interveinal chlorosis and necrosis that resembled the GLSD symptoms of the Esca complex observed in the field. The present study provides the first detailed report of the Esca complex in China. In addition, this is the first record of F. punicata associated with Esca complex of grapevine worldwide. The results of this study provide new insights into the knowledge of the Esca complex.

First Report of Corynespora cassiicola Causing Target Spot on Soybean in Taiwan

Soybean (Glycine max [L.] Merr.) is an important crop in Taiwan. In October 2020, an unknown leaf spot disease was counted (n = 100) to occur over 70% of soybean cultivar ‘Hualien No.1’ in the Shoufeng Township of Hualien County, eastern Taiwan. Initial symptoms on leaves as tiny lesions approximately 3 mm in diameter, which later enlarged and developed into round, irregular, and reddish-brown spots with concentric rings surrounded by a yellowish halo. The symptoms appeared on both young and old leaves, but rarely on the stem or pods. The lesions at the margin of healthy and infected tissues were surface-disinfested in 1% NaOCl for 30 seconds, washed twice in sterilized distilled water, dissected and plated on potato dextrose agar (PDA) to isolate the potential pathogen. Colonies on PDA exhibited light to dark brown color at 24°C with 12-hours light after 7-days incubation. The average growth rate was 3 mm per day. Conidia were light brown in color and obclavate to cylindrical in shape. The size of a conidium was measured with an average of 110.8 ± 28.2 μm in length and 15.2 ± 2.8 μm in width, typically with 3 to 18 septa (n = 50). To confirm the pathogenicity of this fungus, conidial suspension (104 conidia/mL) of two isolates, HL_GM-6 and HL_GM-7, were sprayed on the healthy leaves of 4-weeks-old soybean. Plants sprayed with sterile distilled water were used as a control. After inoculation, the plants were covered with plastic bags to maintain a high humidity for 24 hours before moving into a greenhouse with a condition of 20 to 25°C and relative humidity of 75 to 80%. After 7 days of inoculation, foliar symptoms began to appear and which were identical with the field observations. To complete the Koch’s postulates, pathogen isolation was attempted and the identical fungus was retrieved from the foliar spots of the inoculated leaves. The foliar symptoms as well as the morphology of the conidiophores and conidia suggested the pathogen to be Corynespora cassiicola (Ellis et al. 1971). Molecular characterization was performed using the sequences of internal transcribed spacer (ITS) region of rDNA, actin (act1), tubulin, and translation elongation factor 1 alpha (tef1) genes after a PCR with ITS1/ITS4 (White et al. 1990), ACT-512F/ACT-783R (Carbone and Kohn, 1999), BT2a/Bt2b (Udayanga et al. 2012), EF1-728F/EF1-986R (Udayanga et al. 2012), respectively. BLASTN sequence analyses of the ITS, act1, tubulin, and tef1 genomic regions of the isolate HL_GM-7 (GenBanK accessions MW548097 MW961420, MW961419 and MW961421) showed high similarity with the isolates of C. cassiicola including 99.58% with sequence KF810854 (Deon et al. 2014), 99.11% with FJ853005 (Dixon et al. 2009), 99.34% with MH763700 (Duan et al. 2019), and 99.33% with KY112719 (Zhang et al. 2018) respectively. Based on the morphology, pathogenicity, and sequence results, this study becomes the first report of C. cassiicola causing target spot on soybean in Taiwan. C. cassiicola is known to infect a broad host range (Dixon et al. 2009; Lopezet al. 2018), and it has been found to infect tomato, cucumber, papaya, and Salvia miltiorrhiza in Taiwan (Lu et al. 2019; Tsai et al. 2015). Therefore, the emergence of soybean target spot should be aware to avoid potential damage to soybean production in Taiwan.

A Virus Infecting Hibiscus rosa-sinensis Represents an Evolutionary Link Between Cileviruses and Higreviruses

Hibiscus (Hibiscus spp.) are popular ornamental and landscape plants in Hawaii which are susceptible to foliar diseases caused by viruses belonging to the genera Cilevirus and Higrevirus (family Kitaviridae). In this study, a virus infecting H. rosa-sinensis plants displaying foliar symptoms consistent with infection by a kitavirus, including yellow chlorotic blotches with a green perimeter, was characterized. The genome consisted of two RNAs 8.4 and 4.4 kb in length, and was organized most similarly to cileviruses, but with important distinctions. These included the location of the p29 homolog as the 3′-terminal open reading frame (ORF) of RNA2 instead of its typical locus at the 3′-end of RNA1; the absence of a p15 homolog on RNA2 and the adjacent intergenic region which also harbors small putative ORFs of unknown function; and the presence of an ORF encoding a 10 kDa protein at the 3′-terminal end of RNA1 that was also found to be present in the hibiscus green spot virus 2 genome. Spherical particles approximately 55–65 nm in diameter were observed in infected leaf tissue, and viral RNA was detected by reverse-transcription PCR in individual mites collected from symptomatic plants tentatively identified as Brevipalpus yothersi. Although phylogenetic analyses placed this virus between the higrevirus and cilevirus clades, we propose the tentative taxonomic placement of this virus, designated hibiscus yellow blotch virus (HYBV), within the genus Cilevirus.

Incidence and distribution of Sweetpotato viruses and their implication on sweetpotato seed system in Malawi

A survey was carried out in 19 districts to investigate the prevalence and distribution of sweetpotato virus disease (SPVD) and its implication on the sustainability of clean seed system in Malawi. A total of 166 leaf samples were collected and tested for the presence of 8 viruses using nitrocellulose membrane enzyme-linked immunosorbent assay (NCM-ELISA). SPVD foliar symptoms were observed in 68.42% of the surveyed districts. There were significant variations in disease incidence and severity (p < 0.001) among districts, with the highest incidence in Mulanje (28.34%). Average SPVD severity score was 3.05. NCM-ELISA detected sweet potato feathery mottle virus (SPFMV, 30.54%), sweet potato mild mottle virus (SPMMV, 31.14%), sweet potato mild speckling virus (SPMSV, 16.17%), sweet potato C-6 virus (SPC6V, 13.77%), sweet potato chlorotic stunt virus (SPCSV, 22.16%), sweet potato collusive virus (SPCV, 30.54%), sweet potato virus G (SPVG, 11.38%), cucumber mosaic virus (CMV, 7.78%) either in single or mixed infections. Data from this study indicate a significant SPVD occurrence in the country, and the consequence implications towards national sweetpotato seed system.

The effect of applied salinity and water stress on chemical suppression of Phytophthora ramorum from soilborne inoculum in Rhododendron

A serious concern for nurseries is the potential for Phytophthora ramorum and other Phytophthora species to colonize roots without inducing aboveground symptoms in plants that then serve as cryptic reservoirs of inoculum. Episodic abiotic stresses that reduce plant water potential can compromise host resistance to trigger disease development from root and crown infections in many Phytophthora-plant interactions. We conducted a series of experiments with root-inoculated Rhododendron plants in a potting soil mix to assess influence of excess salt or water deficit on ramorum blight development and the potential for these abiotic stresses to affect efficacy of oomycete-suppressive chemical soil treatments. In growth chamber trials, P. ramorum colonized roots in both non-salted and salt-treated plants. However, salt treatment offset the benefit realized from soil treatment with mefanoxam (Subdue Maxx) or mandipropamid (Micora), as evidenced by enhanced pathogen colonization of roots. A three-week episode of water stress imposed after chemical treatment but prior to inoculation eliminated protection against P. ramorum root colonization conferred by fosetyl-Al (Aliette). In an outdoor experimental nursery, foliar symptoms were apparent in 23% of root-inoculated plants in two trials and absent in one trial. However, the majority of inoculated plants in all trials had colonized roots with little or no aboveground symptoms. A single application of Subdue Maxx or Aliette reduced root colonization by P. ramorum in Rhododendron plants. Although salt stress did not enhance ramorum blight symptom expression in the nursery, salt partially offset protection from P. ramorum root colonization obtained by Subdue Maxx.