Chlorotic Lesions Research Articles

Serological Detection of Cucumber Mosaic Virus and Capsicum Chlorosis Virus in Pepper (Capsicum annuum L.) Germplasm

A survey was conducted on the basis of symptomatology for the screening of germplasm under insect proof glasshouse conditions in the Department of Vegetable Science, Dr. YSPUHF, Nauni, Solan to identify the sources of resistance in pepper crop. The most prominent symptoms were mosaic, mottling, leaf narrowing, stunted growth, mosaic with chlorotic lesions, formation of rings with necrotic lesions and mosaic with mottling. Forty-eight varieties or breeding lines were screened for the presence or absence of virus through DAS-ELISA. The study revealed that nineteen varieties or breeding lines tested positive and twenty-nine tested negative for virus infection. Sweet Banana variety recorded the least O.D. value followed by SB and UHF-Cap-30 whereas, UHF-Cap-22 was most susceptible variety with maximum O.D. value. Mixed infection of CMV and capsicum chlorosis orthotospovirus (CaCV) was recorded with six varieties or breeding lines during germplasm screening. This study signifies the presence of mixed infection with chlorotic lesions like symptoms and importance of DAS-ELISA in screening of pepper germplasm against CMV and CaCV.

First Report of Didymella pomorum Causing Leaf Blight on Angelica sinensis in China.

Angelica sinensis (Oliv.) Diels, is a perennial herbaceous plant of the Umbelliferae family. It has a long history of cultivation and is highly valued as a traditional Chinese medicine in China (Zhang et al. 2012). In September 2023, leaf blight on A. sinensis with an average disease incidence of 56% was recorded in an approximately 6.7-ha production field in Lijiang, Yunnan province, China (26.8215°N, 100.2369°E). At first, small, chlorotic lesions appeared on the leaves. They subsequently increased in density and gradually merged, causing leaves to yellow and wither. Ultimately the blight casused death of the entire foliage. In order to identify the causal agent, cross-sectional segments (5×5 mm2) were cut from the edge of leaf lesions, surface disinfected with a 1% sodium hypochlorite solution for 3 min and rinsed three times with sterile distilled water. They were subsequently placed on potato dextrose agar (PDA) plates and incubated for 3 days under a 12-h photoperiod at 28℃. A total of ten isolates with similar morphological characteristics were obtained by single spore isolation. After 10 days of incubation on PDA, the colony morphology of these isolates was characterized by a brownish central area with a white edge. Aged colonies became wrinkled in the center of the colony. Conidia (n = 30) were elliptical and brown, with a size range of 4.11 to 6.55 μm (average 5.37±0.74 μm) × 3.17 to 4.62 μm (average 3.92±0.43 μm). Chlamydospores (n = 30) formed chains in series, spherical or elliptical in shape, ranging from yellow-brown to dark brown, with a size range of 12.30 to 13.70 μm (average 12.98±0.46 μm) × 4.20 to 5.30 μm (average 4.63±0.26 μm). The nuclear ribosomal internal transcribed spacer region (ITS), the second largest subunit of RNA polymerase II (RPB2), and the 28S nuclear ribosomal large subunit rRNA (LSU) region of two isolates were amplified with the primer pairs ITS1/ITS4 (White et al. 1990), fRPB2-5F/fRPB2-7cR (Liu et al. 1999), and LR0R/LR5 (Schoch et al. 2012), respectively. These amplicons were sequenced bidirectionally and assembled. The two isolates produced the same nucleotide sequences, and the sequences of a representative isolate (AsDp1) were deposited in GenBank. BLASTn analyses showed that the ITS (PP510616), RPB2 (PP526010), and LSU (PP550143) sequences of isolate AsDp1 were 100%, 99.66%, and 100% identical with those of Didymella pomorum ex-type isolate CBS 354.52 (MH857081, KT389616, and MH868616), respectively. A phylogenetic tree was constructed based on the ITS, RPB2, and LSU concatenated nucleotide sequences using the maximum likelihood method in MEGAX. Isolate AsDp1 was clustered with four D. pomorum isolates. According to the morphological and nucleotide sequences analyses, isolate AsDp1 was identified as D. pomorum (Chen et al. 2015). To determine pathogenicity, 1-year-old A. sinensis plants (approximately 20 cm tall) grown in 7-liter pots filled with sterilized field soil were sprayed until runoff with a 1×106 conidia/ml suspension of isolate AsDp1 onto the foliage, while control plants were sprayed with sterile water. All plants were cultivated under a 12-h photoperiod at 25℃. The pathogenicity tests were performed in triplicate with ten plants in each treatment. After fifteen days, numerous chlorotic lesions appeared on the leaves of all inoculated plants. The symptoms were similar to those found on naturally infected plants in the field, while the control plants remained asymptomatic. Subsequently, D. pomorum was reisolated from the diseased leaves, and the identity was confirmed based on its ITS sequence and morphological characteristics. D. pomorum causing stem canker on Rosa spp. was reported in Canada (Ilyukhin 2022). To our knowledge, this is the first report of D. pomorum causing leaf blight on A. sinensis in China. This etiological finding will potentially pave the way for the development of control strategies of this disease.

Isolation and identification of Cladosporium tenuissimum causing leaf blight on garden pea (Pisum sativum L.) in Telangana from southern India

Garden pea (Pisum sativum L.), is an important legume grown worldwide as a source of proteins and carbohydrates. Since pea crop is cultivated globally in cool temperate climatic conditions, increasing temperatures accompanied by high humidity favors fungal infections. A fungal infection causing leaf blight symptoms was frequently observed on the leaves of pea plants grown in greenhouse and field conditions. The blight symptoms showed a water-soaked and wilting appearance with curly leaf edges accompanied by greyish mycelial growth and chlorotic lesions. The disease incidence in field conditions ranged between 5 and 10 %, whereas in the controlled greenhouse conditions, it ranged from 40 to 45%. Two isolates of the fungus, Gp03 and Gp04 were isolated from Arkel pea leaves and purified from a single conidium each. According to Koch's postulates, the pathogenicity tests with these isolates using detached leaf assay and whole plant leaf assays confirmed the pathogen. Morphological and molecular analysis of nucleotide sequences of the D1-D2 region, translation elongation factor-1α (TEF) and actin (ACT) genes confirmed these isolates as members of the genus, Cladosporium. The phylogenetic relatedness of these isolates with other members of the Cladosporium genus revealed them as fungal strains of Cladosporium tenuissimum. Further, these isolates were also confirmed as C. tenuissimum by Microbial Type Culture Collection and Gene Bank (MTCC), Chandigarh in India, and provided the accession nos. MTCC 13581 and MTCC 13582 to the C. tenuissimum strains, Gp03 and Gp04 respectively. This is the first report of Cladosporium tenuissimum infecting garden pea in Telangana, India.

Molecular Characterization and Pathogenicity of Groundnut Bud Necrosis Virus (Orthotospovirus arachinecrosis) Inducing Necrosis in Black Gram

Orthotospovirus arachinecrosis commonly known as Groundnut bud necrosis virus (GBNV), is the causal agent for necrosis disease in black gram. GBNV is the second most important challenge to black gram production in major growing areas following yellow mosaic disease. Symptoms of necrosis disease of black gram include severe downward curling and twisting of leaf lamina, veinal necrosis, petiole necrosis, bud necrosis, and necrotic streaks on the stem. Infection at early growth stages of black gram leads to plant death, while surviving plants exhibited stunted growth with a significant reduction in pod yield. The GBNV-BG isolate was maintained in pure form on cowpea cv. C-152, resulted in chlorotic and necrotic local lesions on primary leaves at seven days post-inoculation. The pathogenicity of GBNV on black gram (LBG-645) was further confirmed by artificial sap inoculation. Amplification of the coat protein gene of GBNV was confirmed through RT-PCR using gene-specific primers, producing an amplicon of 800 bp. Sequence analysis of the coat protein gene revealed that the present study isolates OQ683310 and OR295403 shared sequence identity of 98.0 % and 99.3 % respectively at the nucleotide level, with other isolates in the NCBI database. The phylogenetic analysis of the coat protein gene revealed that the isolates under this study were closely linked with the GBNV tomato isolate from Tamil Nadu (MN718651 and MZ505083).

Orthotospovirus iridimaculaflavi (iris yellow spot virus): An emerging threat to onion cultivation and its transmission by Thrips tabaci in India

The yellow spot disease caused by the virus species Orthotospovirus iridimaculaflavi (Iris yellow spot virus-IYSV), belonging to the genus Orthotospovirus, the family Tospoviridae, order Bunyavirales and transmitted by Thrips tabaci Lindeman. At present, emerging as a major threat in onion (Allium cepa) in Tamil Nadu, India. The yellow spot disease incidence was found to be 53–73 % in six districts out of eight major onion-growing districts surveyed in Tamil Nadu during 2021–2023. Among the onion cultivars surveyed, the cultivar CO 5 was the most susceptible to IYSV. The population of thrips was nearly 5–9/plant during vegetative and flowering stages. The thrips infestation was 34–60 %. The tospovirus involved was confirmed as IYSV through DAS-ELISA, followed by molecular confirmation through RT-PCR using the nucleocapsid (N) gene. The predominant thrips species present in onion crops throughout the growing seasons was confirmed as Thrips tabaci based on the nucleotide sequence of the MtCOI gene. The mechanical inoculation of IYSV in different hosts viz., Vigna unguiculata, Gomphrena globosa, Chenopodium amaranticolor, Chenopodium quinoa and Nicotiana benthamiana resulted in chlorotic and necrotic lesion symptoms. The electron microscopic studies with partially purified sap from onion lesions revealed the presence of spherical to pleomorphic particles measuring 100–230 nm diameter. The transmission of IYSV was successful with viruliferous adult Thrips tabaci in cowpea (Cv. CO7), which matured from 1st instar larva fed on infected cowpea leaves (24 h AAP). Small brown necrotic symptoms were produced on inoculated plants after an interval of four weeks. The settling preference of non-viruliferous and viruliferous T. tabaci towards healthy and infected onion leaves resulted in the increased preference of non-viruliferous thrips towards infected (onion-61.33 % and viruliferous thrips towards healthy onion leaves (75.33 %). The study isolates shared 99–100 % identity at a nucleotide and amino acid level with Indian isolates of IYSV in the N gene. The multiple alignment of the amino acid sequence of the N gene of IYSV isolates collected from different locations and IYSV isolates from the database revealed amino acid substitution in the isolate ITPR4. All the IYSV isolates from India exhibited characteristic amino acid substitution of serine at the 6th position in the place of threonine in the isolates from Australia, Japan and USA. The phylogenetic analysis revealed the monophyletic origin of the IYSV isolates in India.

First Report of Green Mould on Leaf of Phaseolus vulgaris Caused by Cladosporium tenuissimum in Liaoning, China.

Phaseolus vulgaris Linn. is a widely cultivated vegetable throughout the world. From spring 2019 to 2022, green mould symptoms were observed on leaves of P. vulgaris in the greenhouse in Liaoning, China, with disease incidence of 8-75% (plants) and 6-23% (leaves). Symptoms appeared as chlorotic lesions covered with dark green mould. The infections started at the apex or margin of the leaves and then spread inward with a characteristic "V" shape. Lesions exhibited curly morphology. 15 leaf samples with typical symptoms were collected from 5 different greenhouses. A total of 75 (5 replicates of each sample) leaf tissues (0.5 cm × 0.5 cm) were selected from the boundary between diseased and healthy parts. These samples were surface sterilized in 0.5% NaClO formin, rinsed 3 times in sterile distilled water and subsequently incubated at 28℃ on potato dextrose agar (PDA) supplemented with streptomycin (50 μg/ml). Numerous morphologically uniform colonies had been purified, with no other fungi observed. Afterwards, the strains were subcultured on malt extract agar (MEA). Colonies on MEA reached 70 to 80 mm diam after 14 days, smoke-grey to pale olivaceous-grey, woolly, sometimes radially wrinkled. The mycelia were pale olivaceous-grey, with hyphae measuring 1-5 μm wide (n = 20). The conidiophores were solitary or in groups of 2 to 5, and measured 50-280(-350) × 2.5-4 μm (n = 20), with 2-7 septa. The conidiogenous cells exhibited a cylindrical-oblong morphology and measured 10-44 × 5 μm (n = 20), with 0-2 septa, and the loci frequently thickened. The conidia were catenate in densely branched chains, ellipsoid to obovoid, smooth, and measured 2.5-5 × 2-3 μm (n = 50), with 0-4 septa. The morphological characteristics were similar to Cladosporium tenuissimum (Zhang 2003). The representative isolate KZ-19 was selected for molecular identification. The rDNA-ITS, translation elongation factor 1-α and actin genes were amplified, sequenced, and the resulting sequence data were submitted to GenBank (ITS: OQ931048; EF-1α: OQ954495; ACT: OQ954496). The BLAST results exhibited a 99 to 100% similarity with the sequences of C. tenuissimum type strain CBS 125995(ITS: HM148197; EF-1α: HM148442; ACT: HM148687). Furthermore, a multi-locus phylogenetic tree was constructed using the PhyloSuite (v 1. 2. 2) software, which revealed that the strains were most closely related to C. tenuissimum (Zhang et al. 2020). Based on both morphological and molecular characteristics, KZ-19 was finally identified as C. tenuissimum (Bensch 2012). Pathogenicity testing was performed on healthy 1-month-old P. vulgaris plants by inoculating the spore suspension (1×106 conidia/ml) of KZ-19 onto leaf surfaces, while control plants were simulated inoculated with sterile water, and five pots were used for each treatment. The test was performed under field conditions of 16-28°C (temperature) and 24-56% (relative humidity). Chlorotic lesions became evident within 2 days of inoculation, followed by the appearance of green mold on leaves after 7 days. No symptoms were observed in the control group. To fulfill Koch's postulates, the pathogen was re-isolated from three inoculated leaves. The morphological identification of re-isolated pathogens was similar to that of originally isolated pathogens. No infection was observed in non-inoculated control. To the best of our knowledge, this is the first report of C. tenuissimum causing green mould on P. vulgaris. As a ubiquitous saprobic hyphomycete, C. tenuissimum has been implicated in leaf mold in Punica granatum and Trifolium repens, larch bud blight, and strawberry blossom blight in previous years (He et al. 1987; Zhang et al. 2003; Zheng et al. 2010; Nam et al. 2015), presenting a potential threat to numerous crops. Therefore, an investigation of its distribution and pathogenic potential is essential in addition to the development of effective disease management strategies.

The Combination of Low-Cost, Red-Green-Blue (RGB) Image Analysis and Machine Learning to Screen for Barley Plant Resistance to Net Blotch.

Challenges of climate change and growth population are exacerbated by noticeable environmental changes, which can increase the range of plant diseases, for instance, net blotch (NB), a foliar disease which significantly decreases barley (Hordeum vulgare L.) grain yield and quality. A resistant germplasm is usually identified through visual observation and the scoring of disease symptoms; however, this is subjective and time-consuming. Thus, automated, non-destructive, and low-cost disease-scoring approaches are highly relevant to barley breeding. This study presents a novel screening method for evaluating NB severity in barley. The proposed method uses an automated RGB imaging system, together with machine learning, to evaluate different symptoms and the severity of NB. The study was performed on three barley cultivars with distinct levels of resistance to NB (resistant, moderately resistant, and susceptible). The tested approach showed mean precision of 99% for various categories of NB severity (chlorotic, necrotic, and fungal lesions, along with leaf tip necrosis). The results demonstrate that the proposed method could be effective in assessing NB from barley leaves and specifying the level of NB severity; this type of information could be pivotal to precise selection for NB resistance in barley breeding.

Deficiencies in cluster-2 ALA lipid flippases result in salicylic acid-dependent growth reductions.

P4 ATPases (i.e., lipid flippases) are eukaryotic enzymes that transport lipids across membrane bilayers. In plants, P4 ATPases are named Aminophospholipid ATPases (ALAs) and are organized into five phylogenetic clusters. Here we generated an Arabidopsis mutant lacking all five cluster-2 ALAs (ala8/9/10/11/12), which is the most highly expressed ALA subgroup in vegetative tissues. Plants harboring the quintuple knockout (KO) show rosettes that are 2.2-fold smaller and display chlorotic lesions. A similar but less severe phenotype was observed in an ala10/11 double KO. The growth and lesion phenotypes of ala8/9/10/11/12 mutants were reversed by expressing a NahG transgene, which encodes an enzyme that degrades salicylic acid (SA). A role for SA in promoting the lesion phenotype was further supported by quantitative PCR assays showing increased mRNA abundance for an SA-biosynthesis gene ISOCHORISMATE SYNTHASE 1 (ICS1) and two SA-responsive genes PATHOGENESIS-RELATED GENE 1 (PR1) and PR2. Lesion phenotypes were also reversed by growing plants in liquid media containing either low calcium (~0.1 mM) or high nitrogen concentrations (~24 mM), which are conditions known to suppress SA-dependent autoimmunity. Yeast-based fluorescent lipid uptake assays revealed that ALA10 and ALA11 display overlapping substrate specificities, including the transport of LysoPC signaling lipids. Together, these results establish that the biochemical functions of ALA8-12 are at least partially overlapping, and that deficiencies in cluster-2 ALAs result in an SA-dependent autoimmunity phenotype that has not been observed for flippase mutants with deficiencies in other ALA clusters.

First Report of Pitaya Virus X Infecting Lophocereus schottii f. mieckleyanus (Thin-Stemmed Totem Pole Cactus) in the United States.

Lophocereus is a genus of three species of columnar cacti native to Arizona and Mexico (Lodi, 2015). These cacti produce several tall, ascending, columnar stems that branch at the base in a candelabra-like arrangement. The most common species, L. schottii is known as the senita cactus. Several unusual knobby-stemmed spineless forms of senita cactus have been found in nature in Baja California, Mexico, which are collectively known as totem pole cacti. The thin-stemmed totem pole cactus, L. schottii f. mieckleyanus is an important part of landscapes in southern Arizona. Cacti are clonally propagated which makes viral infections of economic importance in the ornamental/nursery industry. In February 2023, virus-like symptoms, such as mosaic and chlorotic spots were observed on the stems of L. schottii f. mieckleyanus grown in a nursery in Phoenix, AZ, USA. Total RNA was extracted from two symptomatic cacti (YPHC-61 A & B) following the protocol by Tzanetakis et al. (2007), and cDNA was synthesized using the Superscript IV Reverse Transcriptase (Invitrogen, Vilnius, Lithuania). Reverse transcription polymerase chain reaction (RT-PCR) performed with cactus virus X specific primers (Kim et al. 2016) targeting the coat protein (CP) gene failed to generate any amplicon, while potexvirus-replicase primers, Potex 2RC and Potex 5 (van der Vlugt and Berendsen 2002) targeting RNA-dependent RNA polymerase (RdRp) gene amplified an expected amplicon of ~580 bp from both the samples. One of the amplicons was Sanger sequenced and showed 90.7% nucleotide (nt) identity with pitaya virus X (PiVX) in the GenBank (MN982522). Sequence was submitted in the GenBank under the accession number OR425049. PiVX is a new species of the genus Potexvirus and is named after its origin from pitaya (Hylocereus spp.). Further, RT-PCR was conducted with PiVX-specific primers, CP 110F/CP 604R targeting CP gene (Bae and Park 2022) and RdRp gene (RdRp F 5' GCGTGGGCCCTGGAAAA-3'/RdRp R 5' CTAAGATTCATCAATTCACCTCTCC-3') (this study). Amplicons of ~500 and 1100 bp were obtained using primers, CP 110F/CP 604R and RdRp F/RdRp R, respectively. A BLAST search revealed 90.5% nt identity to PiVX CP sequences (OM802135 and OM802134) and 87.3% nt identity to RdRp sequences (MN982523 and LC654699) in the GenBank. Sequences of isolates YPHC-61A and YPHC-61B were submitted in the GenBank under accession numbers, OQ915350 and PP182358 (CP gene) and OQ915351 and PP209539 (RdRp gene). Phylogenetic analysis based on the combined sequence datasets of CP and RdRp genes also grouped YPHC-61A and YPHC-61B with PiVX isolates and separated from other potexviruses species. For a bioassay of the virus, sap extract from symptomatic cactus was mechanically inoculated onto indicator plant species, i.e., beans, alfalfa, and melon. Ten days post- inoculation, chlorotic lesions were observed on beans and alfalfa plants, while melon and mock-inoculated plants did not show any symptoms. Similarly, L. schottii f. mieckleyanus plants grafted with infected cactus showed chlorotic spots after 30 days post grafting. Mechanically inoculated beans, alfalfa, and cactus plants were found to be positive for PiVX based on RT-PCR and Sanger sequencing. PiVX has earlier been detected on Notocactus leninghausii f. cristatus (Park et al. 2018) and dragon fruit (Selenicereus undatus) plants in South Korea (Bae and Park 2022). To our knowledge, this is the first report of PiVX on L. schottii f. mieckleyanus in the United States and worldwide.

A natural single‐nucleotide polymorphism in the CAMTA3 transcription factor regulates its function and transcription of its target genes

AbstractCAMTA3, a Ca2+/calmodulin‐binding transcription factor, is a key regulator of plant immunity in Arabidopsis. Here, we identified a novel naturally occurring single‐nucleotide polymorphism that results in a missense nonconservative mutation (CAMTA3H386D) in many Arabidopsis ecotypes. This region of CAMTA3 is not part of any previously characterized regulatory domains. To study the consequence of this change on the function of CAMTA3, we introduced the CAMTA3H386D into camta3, a loss‐of‐function mutant that exhibits a constitutive cell death phenotype, chlorotic lesions on leaves, and reduced plant size. Phenotypic and molecular analysis of these lines indicated that the expression of CAMTA3H386D in the camta3 mutant did not complement the mutant phenotypes. Also, the ecotypes containing the CAMTA3H386D exhibited camta3 phenotypes. Marker genes associated with salicylic acid biosynthesis and pathogen response were upregulated in the CAMTA3H386D lines and the Arabidopsis accessions 7127 (Est‐1) and 9941 (Fei‐0), as in camta3, indicating that H386D mutation alters CAMTA3 activity in regulating the expression of known target genes. In Nicotiana benthamiana transient expression assays, CAMTA3H386D failed to induce the expression of a luciferase reporter gene driven by the rapid stress‐responsive elements (RSRE) that contain the known binding sites of CAMTA3, suggesting that CAMTA3H386D mutation impairs its ability to activate its target genes. Transgenic lines and tested natural accessions expressing CAMTA3H386D showed enhanced levels of H2O2 and increased resistance to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. Collectively, our results indicate that the H386D mutation in a previously unknown regulatory region of CAMTA3 is essential for its function.

First Occurrence of Coffee Leaf Rust Caused by Hemileia vastatrix on Coffee in Saudi Arabia

Coffee leaf rust (CLR) is a major disease of Arabica coffee caused by the biotrophic fungus Hemileia vastatrix. Jazan region in Saudi Arabia has long been one of the last coffee-producing regions in the world free of this disease. In August 2023, CLR was initially observed in coffee plantations located in Fyfa district one of the main coffee-producing mountains of Jazan region. The source of the infection is unknown, however CLR could have entered Jazan from the nearest coffee-producing locations that haven the pathogen. During a scheduled survey in August 2023, symptoms were observed including yellowish-orange lesions that frequently combined to form chlorotic lesions and exhibited the powdery appearance of yellow uredospores on the abaxial surface of leaves. The uredospores and teliospores were microscopically examined and their morphology matched the previously published description for H. vastatrix. The identity of H. vastatrix specimens was further confirmed based on PCR amplification and sequencing of ITS, sharing a 99–100% identity with previously published sequences, as belonging to H. vastatrix. The pathogenicity of H. vastatrix specimens was investigated on Coffee arabica plants under growth chamber conditions, and all were pathogenic relative to control, with 100% of disease incidence, therefore fulfilling Koch’s postulates. Based on our findings, this is the first documentation of H. vastatrix causing CLR in Saudi Arabia.

Natural occurrence of pelargonium flower break virus infectingPelargonium zonaleplants in South America

AbstractIn 2023, virus‐like symptoms were observed inPelargonium zonaleplants in Brazil. Transmission electron microscope analysis revealed the presence of isometric particles measuring ca. 30 nm in diameter in leaf extracts and ultrathin sections of symptomatic field plants. Molecular assays using PFBV‐specific primers and sequencing of amplicons identified the pelargonium flower break virus (PFBV, genusAlphacarmovirus). Mechanically inoculated plants ofChenopodium quinoaandP. zonaleexhibited symptoms of chlorotic local lesions and systemic chlorotic spots, respectively. This is the first report of PFBV infectingP. zonaleplants in South America (Brazil).

Pantoea vagans causing soft rot disease in Agave angustifolia, in Mexico

AbstractIn Mexico, agave (Agave angustifolia Haw.) crops stand out regionally due to their socioeconomic, agroecological and cultural importance. It is the most important raw material in the elaboration of two relevant alcoholic beverages: tequila and mezcal. Nevertheless, this crop is susceptible to phytopathological threats mainly fungi and bacteria, both having a massive impact in yearly production. In this paper, we describe an agave disease consisting in reddish chlorotic lesions along the leaf bases resulting in tissue degradation and producing soft rot (softening tissue and firmness loss). Fast‐growing yellow and mucoid bacteria colonies were isolated from the lesions in BK agar. Microscopic analysis resulted in a Gram‐negative bacilli. Pathogenic tests resulted showed identical symptoms to the initial ones in natural maguey host plants as well as in the selected model Aloe vera L. showing more aggressive soft rot symptoms. It is important to highlight that in the selected plant model (Aloe vera), the symptoms arose faster and more aggressively. Biochemical and molecular analysis of the bacterial isolate identifies it as Pantoea vagans, a species not previously documented in this crop. Although all bacterial infections in plants are treated in similar ways, accurate identification could translate into targeted prevention. The detection of Pantoea vagans is considered a relevant problem given the symptoms it causes. We conclusively reported a 37% infection incidence rate, which can mean significant economic losses. However, the role of the genus Pantoea in agave rot has been poorly studied. Therefore, this report shows the identification of a new Pantoea species bounded to agave cultivars in Malinalco municipality, state of Mexico.

First report of bacterial leaf spot caused by Pseudomonas cichorii on Monstera adansonii in Hawai'i, USA.

Monstera adansonii is a popular ornamental house plant prized for its small size and unique leaf fenestrations. In Hawai'i, it is also sold as cut foliage (combined value ~$21K; USDA NASS 2019). In January 2022, yellow chlorotic lesions that progressed to greyish-black and, finally, to brown necrotic lesions were observed primarily along the margins of fenestrations on M. adansonii foliage at a plant nursery in Hilo, HI. All 100 variegated specimens in 4-inch pots were infected, exhibiting symptoms along the lighter yellowish-white margins. The green, unvariegated variety planted along a fence for cut foliage exhibited an infection rate of 10%. Symptomatic leaf tissue was disinfected for 1 minute in a 10% bleach solution. Tissue from the margins of leaf spots was subsequently dissected, soaked in sterile distilled water for 1 hour, and plated on Luria-Bertani (LB) agar. Plates contained nearly pure cream-colored bacterial colonies with undulate margins. Isolates were established from single colonies. One isolate (BCB001) was transferred to King's medium B (KMB) and culture fluorescence was observed under 365 nm UV light. Isolate BCB001, which was gram-negative, was identified as Pseudomonas cichorii based on the LOPAT scheme (Schaad et al. 2001). A partial 16S rRNA gene product (495 bp) using primers Y1/Y3 (Cruz et al. 2001) was sequenced and compared in GenBank (accession no. OQ875210) and was 100% identical to multiple accessions of P. cichorii in the NCBI database. Bacterial identity was further confirmed using the P. cichorii-specific primers Hrp1a/Hrp2a (Cottyn et al. 2011) to amplify and sequence a 790 bp fragment (accession no. OQ850761), which was identical to accession no. MH396007, a P. cichorii isolate recovered from Thai basil in Hawai'i. To prove pathogenicity, strain BCB001 was grown on LB agar for 48 h at 27°C and suspended in sterile water at 107-108 CFU/ml. Four healthy, 2-month-old unvariegated M. adansonii plants produced from cuttings were syringe inoculated following the protocol of Wang et al. (2022). Leaves were injected with sterile water using the same methods and acted as negative controls. Plants were placed in clear plastic bags and held at 24°C with 12 h light for 48 hours in a growth chamber, after which time the plants were removed from the bags and incubated under the same conditions for the remainder of the experiment. Leaf spots were not present on any of the control leaves or on noninjected leaves of the plants after five days of incubation. Grey to black, water-soaked leaf spots 0.84 - 15.24 mm in diameter were present on all injected leaves (96% of the injection sites) 2 days post-inoculation (DPI), which were identical to the original diseased samples. At 5 DPI, spots became dark brown to black with a yellow halo, and the affected tissue was completely collapsed. Bacterial colonies were consistently re-isolated from the lesion margins of inoculated plants and morphologically (LB and KMB) and molecularly (Hrp) identified as P. cichorii, thus fulfilling Koch's postulates. To the best of our knowledge, this is the first report of bacterial leaf spot caused by P. cichorii on M. adansonii in Hawai'i. Since M. adansonii is an ornamental plant that is prized for its leaves, leaf spots caused by P. cichorii can reduce the marketability of inventory. To avoid further spread, increasing plant spacing to improving airflow, decreasing the amount of watering, avoiding mist irrigation, and carefully removing and discarding diseased leaves are suggested.

Global transcriptomic analysis in avocado nursery trees reveals differential gene expression during asymptomatic infection by avocado sunblotch viroid (ASBVd)

Avocado sunblotch viroid (ASBVd) is the type species of the family Avsunviroidae and the causal agent of avocado sunblotch disease. The disease is characterised by the presence of chlorotic lesions on avocado fruit, leaves and/or stems. Infected trees may remain without chlorosis for extended periods of time, though distorted growth and reduced yield has been observed in these cases. The molecular effects of ASBVd on avocado, and members of the Avsunviroidae on their respective hosts in general, remain poorly understood. Host global transcriptomic studies within the family Pospiviroidae have identified several host pathways that are affected during these plant-pathogen interactions. In this study, we used RNA sequencing to investigate host gene expression in asymptomatic avocado nursery trees infected with ASBVd. Transcriptome data showed that 631 genes were differentially expressed, 63 % of which were upregulated during infection. Plant defence responses, phytohormone networks, gene expression pathways, secondary metabolism, cellular transport as well as protein modification and degradation were all significantly affected by ASBVd infection. This work represents the first global gene expression study of ASBVd-infected avocado, and the transcriptional reprogramming observed during this asymptomatic infection improves our understanding of the molecular interactions underlying broader avsunviroid-host interactions.

First report of konjac mosaic virus in Spuriopimpinella brachycarpa in Korea.

Spuriopimpinella brachycarpa Nakai (Common name, Chamnamul; family Apiaceae) is a plant whose leaves are consumed as a vegetable and used as a folk medicine in Korea (Kim et al., 2020). In February 2020, seven samples of S. brachycarpa leaf showing virus symptoms including yellowing, vein chlorosis, chlorotic lesions, and severe mottling were collected from a greenhouse in Busan, South Korea, to diagnose the potential disease (Fig. S1a, b). The disease incidence rate in the greenhouse was >10% (2,970 m2). To identify the causal virus, we analyzed leaf dip preparation and thin sections of the symptomatic leaves by transmission electron microscopy. Filamentous virus particles and pinwheel structures were observed, indicating the presence of a potyvirus (Fig. S1c, d). To confirm these results, the symptomatic leaf samples were further analyzed by reverse-transcription polymerase chain reaction (RT-PCR) using potyvirus universal primers (Table S2) and direct sequencing of the PCR products. All samples were positive for konjac mosaic virus (KoMV). To exclude the possibility of infection by multiple viruses, we performed high-throughput sequencing (HTS) on an Illumina NovaSeq 6000 system (Macrogen Inc., Seoul, South Korea). There were two contigs (9,267 and 2,851 nt) mapping to KoMV sequences. A large contig (9,267 nt; 705,967 mapped reads; mean read coverage of 11,351.4x) showed about 80% identity (93% coverage) with KoMV-F (GenBank accession no. NC_007913) isolated from Amorphophallus konjac in Japan (Nishiguchi et al., 2006). To isolate KoMV from S. brachycarpa, we mechanically inoculated leaf extracts from symptomatic samples onto Chenopodium quinoa as an assay host via three single-lesion passages, followed by propagation in Nicotiana benthamiana. In a bioassay of the KoMV isolate (KoMV-BS), we mechanically inoculated sap from infected N. benthamiana onto 31 indicator plants including Cryptotaenia japonica (Apiaceae), which is similar to S. brachycarpa (Table S3). KoMV-BS systemically induced vein chlorosis and/or leaf mottling in four Nicotiana species and C. japonica, and chlorotic local lesions in upper leaves of C. quinoa; no symptoms were observed in 25 other indicator plants. These results were confirmed by RT-PCR. Next, we obtained the complete genome sequence of KoMV-BS using HTS and 5' and 3' rapid amplification of cDNA ends, with newly designed primers (Table S2). The assembled full-length KoMV-BS genome sequence was 9,392 nt in length, excluding the poly(A) tail, and encoded a polyprotein composed of 3,060 amino acids. The sequence was deposited in GenBank (accession no. OR001914). BLAST analysis showed 84~88% and 90~98% identities at CP nucleotide and amino acid levels, respectively with the reported KoMV isolates, confirming the virus to be an isolate of KoMV (synonym; Japanese hornwort mosaic virus, zantedeschia mosaic virus) (Adams et al., 2005; Nishiguchi et al., 2006). KoMV infection was first reported in A. konjac from Japan (Shimoyama et al. 1992) and has been spread worldwide as one of the major causal agents of viral diseases in calla lily (Liao et al., 2020). To the best of our knowledge, this is the first report of KoMV infection in S. brachycarpa. To date, cucumber mosaic virus and tobacco mosaic virus have been reported to infect S. brachycarpa in Korea (Yoon et al., 2016; 2017). Our findings will be helpful for developing virus-management strategies to prevent yield and quality loss in S. brachycarpa.

First Report of Stemphylium lycopersici Causing Leaf spot on Sedum plumbizincicola in Hunan Province of China.

Sedum plumbizincicola is a perennial succulent herb that can hyperaccumulate high concentrations of cadmium and zinc (Liu et al. 2017). In October 2021, a leaf spot disease occurred on S. plumbizincicola seedlings in a nursery in Changsha (28°13' N; 112°56'E), the Hunan Province of China. Almost 30% of the nearly 1 million seedlings were infected. Symptoms initially appeared as small brown spots on the leaf surface or edges, gradually enlarged, becoming oval, and bearing chlorotic lesions with dark brown borders. Eventually, the center of the lesions became sunken and then fell off. Eight symptomatic plant samples were collected by five-point sampling method (Zheng et al. 2018). Small pieces of 5×5 mm were excised from the lesion margins, sterilized with 70% ethanol for 10 s, 0.1% HgCl2 for 40 s, rinsed with sterile distilled water three times, and then cultured on potato dextrose agar (PDA) at 26 °C for 5 days in the dark. Fungal colonies showing similar morphology were observed from all the isolated samples and, in total, eight fungal strains were obtained. On PDA, fungal colonies were initially white, and later become light gray. After cultured on V8 juice agar (V8A, each litre of medium contains 200 mL of V8 juice, 3 g of CaCO3 and 15 g of agarose) for 14 days (Hyowon et al. 2016), conidia of a representative isolate SY-1 were produced, which were oblong, muriform, with blunt ends and conical apex, pale to light brown, and constricted at the 1 to 3 major transverse septa, 38.34-46.68 μm×11.67-18.34 μm (n=50). These morphological characteristics were consistent with that of Stemphylium lycopersici (Nasehi et al. 2016). The internal transcribed spacer (ITS) region of rDNA and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene of representative isolates SY-1 to SY-3 were amplified and sequenced using the primer pairs ITS4/ITS5 and gpd1/gpd2 as described previously (Woudenberg et al. 2017). BLASTn analysis showed that ITS sequences of isolates SY-1, SY-2 and SY-3 (accession nos. OP317641, OQ852042 and OQ852043) had more than 99% identity with Stemphylium sp, while GAPDH sequences (OP331223, OQ858620 and OQ858621) had 100% identity with S. lycopersici KR911813 (Sun et al. 2016). A concatenated ITS-GAPDH phylogenetic tree grouped our isolates within the S. lycopersici clade. For the pathogenicity test, one-month-old potted S. plumbizincicola seedlings were inoculated with conidia suspension (105 conidia/ml), which was induced on V8A. Four sites of each leaf of the potted S. plumbizincicola plants were dropped with a conidia suspension of strain SY-1, with 10 μL per site. Leaves treated with sterile water were served as controls. All of the inoculated seedlings were placed in a growth chamber at 26°C with a photoperiod of 12 h. The pathogenicity tests were repeated twice, with each had three replicative plants. After 7 days, all the inoculated leaves developed brown spots resembling those observed in the nursery, whereas the control plants remained symptomless. Stemphylium lycopersici was specifically re-isolated and identified by morphological and molecular methods (accession nos. OQ852045 for ITS and OQ858622 for GAPDH, respectively), thus fulfilling Koch's postulates. To our knowledge, this is the first report of S. lycopersici causing leaf spot on S. plumbizincicola in China. Since S. plumbizincicola played an important role and widely planted for heavy metal pollution treatment (Jiang et al. 2010), and this disease might seriously influence the S. plumbizincicola seedling breeding, identification of the pathogen might provide a foundation for the diagnosis and control of the disease.

Identification, pathogenicity, and fungicide sensitivity of Eutiarosporella dactylidis associated with leaf blight on maize in China

Maize (Zea mays L.) is an economically vital grain crop that is cultivated worldwide. In 2011, a maize foliar disease was detected in Lingtai and Lintao counties in Gansu Province, China. The characteristic signs and symptoms of this disease include irregular chlorotic lesions on the tips and edges of infected leaves and black punctate fruiting bodies in dead leaf tissues. Given favourable environmental conditions, this disease spread to areas surrounding Gansu. In this study, infected leaves were collected from Gansu and Ningxia Hui Autonomous Region between 2018 and 2020 to identify the disease-causing pathogen. Based on morphological features, pathogenicity tests, and multi-locus phylogenetic analysis involving internal transcribed spacer (ITS), 18S small subunit rDNA (SSU), 28S large subunit rDNA (LSU), translation elongation factor 1-alpha (TEF), and β-tubulin (TUB) sequences, Eutiarosporella dactylidis was identified as the causative pathogen of this newly discovered leaf blight. Furthermore, an in vitro bioassay was conducted on representative strains using six fungicides, and both fludioxonil and carbendazim were found to significantly inhibit the mycelial growth of E. dactylidis. The results of this study provide a reference for the detection and management of Eutiarosporella leaf blight.

First Report of Passiflora latent virus in Passion fruit (Passiflora edulis) in China.

Passion fruit (Passilora edulis, family Passifloraceae.) is an economically important fruit crop in many tropical and subtropical regions worldwide. It is widely planting in southern China, and in greenhouses throughout the country. In Mar 2022, symptoms of a viral-like infection were observed on the leaves of passion fruit plants in a 3-hectare greenhouse complex in Hohhot, China. Chlorotic lesions were observed on leaves of two vines of passion fruit and symptomatic leaves developed chlorotic spots, followed by systemic leaf chlorosis and necrosis. Dark ringed spots emerged on the surface of matured fruits (Figure 1). To confirm infectivity, mechanical transmission of the virus was performed by grinding leaves from two symptomatic passion fruit vines in 0.1M phosphate buffer pH 7, and the resulting two samples were each used to rub-inoculate carborundum-dusted leaves of three healthy passion fruit seedlings. Newly emerging leaves of inoculated plants developed mild mosaic symptoms 30-days after inoculation. Three samples from each of the two original symptomatic plants and two samples from each inoculated seedling tested positive using a Passiflora latent virus (PLV) ELISA Kit (Creative Diagnostics, USA). To further confirm the virus identity, total RNA from leaf samples from one of the original greenhouse symptomatic plants and one of the inoculated seedlings were extracted using an TaKaRa MiniBEST Viral RNA Extraction Kit (Takara, Japan). The two RNA samples were subjected to reverse transcription-polymerase chain reaction (RT-PCR) tests with virus specific primers PLV-F (5'-ACACAAAACTGCGTGTTGGA-3') and PLV-R (5'-CAAGACCCACCTACCTCAGTGTG-3') (Cho et al., 2020). RT-PCR products of the expected 571 bp were obtained from both the original greenhouse sample and inoculated seedling. Amplicons were cloned into the pGEM-T Easy Vector, and two clones per sample were Sanger sequenced bidirectionally (Sangon Biotech, China), and the sequence of one clone from one of the original symptomatic sample was uploaded to NCBI (GenBank OP320922.1). This accession had 98% nucleotide sequence identity with a PLV isolate from Korea (GenBank: LC556232.1). RNA extracts from two asymptomatic samples tested negative for PLV with both ELISA and RT-PCR tests. We also tested the original symptomatic sample for common occurring passion fruit viruses, including passion fruit woodiness virus (PWV), cucumber mosaic virus (CMV), east asian passiflora virus (EAPV), telosma mosaic virus (TeMV), papaya leaf curl Guangdong virus (PaLCuGdV), and the RT-PCR results were negative for those viruses. However, based on the systemic leaf chlorosis and necrosis symptoms, we cannot preclude a mixed infestation of other viruses. PLV affects fruit quality and has high potential to reduce market value. To our knowledge, this is the first report of PLV in China, which could provide a reference basis to PLV identification, prevention and control. Acknowledgments This research was carried out with the support of Inner Mongolia Normal University High-level Talents Scientific Research Startup Project (Grant no. 2020YJRC010). Supplementary material Figure 1. Mottle, leaf distortion, puckering symptoms on old leaf (A), mild puckering symptom on young leaf (B), and ring-striped spots symptoms on fruit (C) of the PLV infected passion fruit plant in China.

Antiviral Efficacy of Bacillus sp. against Groundnut bud necrosis orthotospovirus in Cowpea

Tomato spotted wilt, a disease caused by Groundnut bud necrosis virus (GBNV), is a major disease affecting tomatoes causing great yield loss to the farming community. In this study, the GBNV-TNAU 1 virus isolate was obtained from infected plants and maintained through mechanical transmission on cowpea cv. CO7, which induced chlorotic and necrotic local lesions by the fourth-day post-inoculation (dpi). The pathogenicity of GBNV on tomato cv. PKM1 was confirmed through sap transmission. In vitro screening of Bacillus sp. viz., B. amyloliquefaciens (Ka1), B. subtilis (Bbv 57), and B. subtilis (BST 8) demonstrated effective reduction of GBNV-induced lesions in cowpea. B. amyloliquefaciens (Ka1) and B. subtilis (BST8) displayed a maximum germination percentage of 100 %, while B. amyloliquefaciens (MM12) exhibited 83.33 %. Futhermore, maximum vigour index of 1743.8, 1699.6 & 1436.5 was noticed in B. amyloliquefaciens (Ka1), B. subtilis (BST8) & B. subtilis (Bbv 57) treated plants. These findings provide valuable insights on sustainable management strategies against the disease, benefiting farmers by enhancing tomato cultivation productivity and profitability.