Yellow Spot Research Articles

Solubility tuning of alkyl amine functionalized carbon quantum dots for selective detection of nitroexplosive

The major issue of luminescent carbon quantum dots (CQDs) is the solubility in wide range of solvent and disappearance of luminescence behavior in its solid state due to aggregation caused quenching. To resolve this issue, in the present work, we have tuned the solubility of carbon quantum dots in different kinds of solvents through surface functionalization technique. Here, we have synthesized three sets of alkyl amine functionalized carbon quantum dots from citric acid and alkyl amine by solvothermal method. We have observed that the solubility of CQDs increases from aqueous medium to organic medium with increasing length of hydrophobic alkyl chains attached at the surface of CQDs. It is seen that n-butyl amine functionalized CQDs (C4-CQD) are soluble in any type of solvents. Here we have also observed that the C4-CQD shows aggregation induced emission instead of aggregation caused quenching in solid state and shows bright yellow luminescence color upon excitation. Besides this, we also report selective detection of both dinitroaniline (DNA) and dinitrophenol (DNP) using this C4-CQD via fluorescence quenching and colorimetric methods. In organic medium both shows significant quenching with emission peak of carbon quantum dots is red shifted (∼30 nm) in presence of DNP whereas no significant peak shift is observed for DNA. CQDs containing paper strip shows yellow spot in presence of DNP but no such characteristic color is appeared after addition of DNA. Using these two techniques, we have selectively identified both DNA and DNP among other nitro compounds by our as-synthesized carbon dots.

Molecular Characterization of a Bipartite Begomovirus and an Alphasatellite Associated with Leaf Curl and Mosaic Disease of Bitter Gourd in Punjab

Bitter gourd (Momordica charantia) is an economically important vegetable and medicinal plant that gets infected by a number of viruses. A survey was conducted in and around Jalandhar region of Punjab, India to study viral symptoms in the bitter gourd fields. The plants showed reduced fruit size, upward leaf curling and mosaic symptoms, indicating begomovirus infection. DNA was isolated from the symptomatic leaves and tested for begomovirus infection using group-specific PCR primers. Amplified products were cloned into pMD20-T vector and sequenced. Based on the obtained sequence, new sets of primers were designed and used for amplification of complete DNA-A and DNA-B. Complete DNA-A and DNA-B were cloned in pMD20-T and sequenced. Sequence analysis of the present DNA-A and DNA-B showed their highest identity with Tomato leaf curl New Delhi virus (ToLCNDV). The associated Tomato leaf curl New Delhi alphasatellite was amplified using universal primers, cloned and sequenced. In phylogenetic analysis, the present DNA-A and DNA-B grouped with ToLCNDV isolates and other begomoviruses reported from bitter gourd. Phylogenetic analysis of the present alphasatellite showed its closet identity with Tomato leaf curl New Delhi alphasatellite. Recombination analysis of the DNA-A sequence showed that the present bitter gourd isolate might be a recombinant of Squash leaf curl China virus (SLCChV) as a major parent and Chilli leaf curl virus as a minor parent. Analysis of the DNA-B suggested it to be a recombinant of SLCChV as a major parent and Chyaote enation yellow mosaic virus as a minor parent. The present alphasatellite might be a recombinant of Tomato yellow spot alphasatellite as a major parent and Croton yellow mosaic alphasatellite as a minor parent. The percent identities of the nucleotide and amino acid sequences validate and support the results of the phylogenetic and recombination analyses.

First report of leaf blight caused by Pantoea agglomeranson wheat in China.

Wheat (Triticum aestivum L.) is the main grain crop in Ningxia Hui Autonomous Region, China. A new leaf blight disease of wheat was observed in many wheat fields in Yinchuan City and Wuzhong City of Ningxia during 2020-2021. The average disease incidence of the cultivar Ningchun 50 was 5 to 15%, and there appeared the evident disease symptoms from the heading stage, then the symptoms got more serious until the mature stage. The tips of the leaves were chlorotic and turned bright yellow at the early stage of the disease. Later on, the yellow leaf spots were further spread from the tip to the petiole, and the yellow-colored necrotic lesions emerged, resulted in withering and death of leaves (e-Xtra 1, a-d). To isolate and identify the pathogenic agent, diseased leaves were cut into 0.5 cm × 0.5 cm small pieces, and sterilized in 5% NaOCl solution for 5 min, and were rinsed three times in sterile water, then crushed with tweezers in 2 mL sterilized water and streaked three times onto Nutrient Agar (NA) medium. and 15 single colonies which had the same colony morphology were obtained. Of the 15 colonies, 3 (named WH1, Cx1 and HJ1) were randomly selected for further morphological, biochemical and molecular characterization. The resulting bacterial colonies were incubated at 29±1°C in the dark for 3 days, colony morphology was raised, mucoid texture, round, and smooth with entire margin; the color of these colonies was white at the beginning and turned yellow later. These bacteria were rod-shaped gram-negative cells with peritrichous flagella. Based on the physiological and biochemical assay results (e-Xtra 2), the three strains were initially identified as Pantoea agglomerans (Wang, D H., et al. 2021; Wang, J J., et al. 2021). 16S rDNA and gyrB of the three strains were amplified and sequenced by ABI3730XL sequencer in GENEWIZ (Suzhou, China). The sequences of 16S rDNA and gyrB of these strains were submitted to GenBank with the accession numbers ON428446, ON428461 and ON428462 for 16S rDNA; ON461799, ON461801, ON461803 for gyrB. 16S rDNA and gyrB sequences homology analysis showed that the three strains had the highest homology which were over 99.5% with the sequences of the reported P. agglomerans (e-Xtra 1, g) . A phylogenetic analysis based on 16S rDNA and gyrB gene sequences was performed using the MEGA6.0 proximity method, and the results of the phylogenetic tree showed that strains Cx1, WH1 and HJ1 clustered on the same clade with the reported P. agglomerans strains (e-Xtra 1, h-j). Thus, Cx1, WH1 and HJ1 were identified as P. agglomerans. Pathogenicity test was performed to complete Koch's postulates, Ningchun 50 was planted in pots, four-week-old healthy wheat seedlings were inoculated with 107 CFU/mL bacterial suspension using two inoculation methods: 1) Leaf surface was poked with disposable syringe needle, and 50 µL of suspension was injected into each of the pinholes (Suraj, S., et al. 2020); 2) Leaf was cut at 45° at the lower 2-3 cm of the leaf tip with scissors dipped in the bacterial suspension. Wheat leaves inoculated with sterile distilled water were regarded as controls. The inoculated wheat was cultivated in a greenhouse (temperature 28 ± 2°C, humidity 40 ± 2%) and covered in transparent polyethylene bags at first 96 h. Symptoms appeared at 3 days after inoculation, and after 7 days, the acupunctured wheat leaves turned chlorotic and yellow around the pinholes and some were necrotic; the leaf-cutting wheat turned yellow and necrotic from the clipping point to the leaf base; the acupunctured and cut leaves totally died after 15 days, and all of the control leaves were healthy (e-Xtra 1, e-f). Subsequently, pathogens were reisolated from inoculated leaves, and identified as P. agglomerans according to molecular identification described above. To our knowledge, this is the first report of leaf blight disease of wheat caused by Pantoea agglomeransglobally as well as in China. Identifying the cause of the disease will support efforts for its future control and management.

First Report of Pseudopestalotiopsis theae Causing Leaf Spot on Euonymus japonicus in China.

Euonymus japonicus Thunb., an evergreen shrub, is popular for landscaping in China. In 2021, leaf spot was observed on E. japonicus (about 150 trees) leaves with 40 to 50% disease incidence in Wanzhou urban forest (30°45'N; 108°27'E) of Chongqing, the infected plants were between 5 and 6 years old. The symptoms started to occur from June to July and approximately 30 to 40% of the leaves exhibited leaf spot symptoms from August to September. Initial symptoms appeared as yellow spots of 1.2 to 4.9 mm in diameter, and then expanded to become large and irregular lesions, having white center surrounded by a brown halo. Under humid conditions, black dots appeared in the central part of the spots. In later stage, split and fall of the tissues occurred from the infected spot. To identify the causal agent, infected tissues from 20 samples (from 5 trees) were cut into small pieces (5 mm2), surface-sterilized for 30 s in 75% ethanol and 3 minutes in 3% sodium hypochlorite, rinsed three times in sterile water, placed onto potato dextrose agar (PDA) amended with streptomycin sulfate (50 μg/ml) and incubated at 25°C in dark conditions. Purified eight fungal colonies were white with undulating margins and light cream on the reverse side, measuring 85 mm diameter after 7 days, dark brown to black conidiomata were irregularly scattered and Conidia were observed in 20 days old colonies. Conidia were spindle-shaped, 4.5 to 6.8 × 15.2 to 23.5 μm (n=50), with 4 diaphragms, the three median cells were light to dark brown and the two end cells were colorless. 1 to 3 accessory filaments (5.2 to 22.5 μm long) protrude from theapical cell while a short stalk (3.5 to 5.5 μm long) was attached to the basal cell, these morphological features suggested that the isolates were most likely Pestalotiopsis. sp. Eight colonies were confirmed to be identical based on morphological characteristics. For molecular identification, DNA was extracted from representative strains (YF-5, YF-13, YF-24). The internal transcribed spacer (ITS) region, β-tubulin (TUB2), the translation elongation factor-1 alpha gene (TEF1), genes were amplified using primers ITS5/ITS4, TUB2F/TUB2R, and EF-526F/EF-1567R, respectively (White et al.1990; Glass & Donaldson 1995; O'Donnell & Cigelnik 1997; Carbone &, Kohn.1999). The sequences were deposited in NCBI GenBank YF-24, [ITS; ON204233: TUB2; ON304156: TEF1; ON400075]: YF-5, [ITS; OP379570: TUB2; OP413495: TEF1; OP413496]: YF-13, [ITS; OP379589: TUB2; OP413494: TEF1; OP413497]. Which revealed a 95% similarity to the Ps. theae NTUCC 18-067 [ITS; MT322086: TUB2; MT321888: TEF1; MT321987] ex-type sequences. Based on morphology and multilocus phylogenetic analysis, representative strains were identified as Pseudopestalotiopsis theae. For Koch's postulates, wiped the leaves of six healthy plants of E. japonicus (two-year-old) grown in pots with sterile water, 10 μL of spore suspension (106 spores/ mL) was brushed on five leaves per plant (three plants in total) with a sterile brush, and the other three plants were treated with sterile water instead of spore suspension as control, the plants were placed in a greenhouse at 28°C and 95±1% relative humidity. Seven days after inoculation, brown lesions appeared, similar to those observed in infected plants. Black dots surrounded by a brown halo reappear on the lesions after 12 days, whereas control plants remained healthy. Ps. theae culture was re-isolated from the infected leaves and identified using morphological characteristics and DNA sequence analysis. To our knowledge, Ps. theae can cause diseases on tea plants and has been found in Japan, Thailand and China, this is the first report of leaf spot infection of E. japonicus caused by Ps. theae in China. This disease is reducing the ornamental value of E. japonicus. Our results will contribute to the prevention and cure of leaf spot disease in E. japonicus.

First report of Cotton leafroll dwarf virus infecting cacao (Theobroma cacao L.) trees in Brazil.

Cotton leafroll dwarf virus (genus Polerovirus, family Solemoviridae) has been commonly reported affecting cotton plants (Gossypium spp., family Malvaceae) and several weed species (Ramos-Sobrinho et al., 2021; Sedhain et al., 2021). During a recent survey, cacao (Theobroma cacao L.) trees exhibiting virus-like symptoms such as leaf mosaic, vein clearing, and yellow spot were observed in the south part of the state of Bahia, northeastern Brazil, in 2022. Leaf samples were randomly collected from symptomatic cacao plants (n=30) growing in an affected area of approximately 30 ha. Total RNA obtained from pooled cacao samples were subjected to Illumina HiSeq 2500 sequencing as previously described (Keith et al., 2021), and partial sequences of cotton leafroll dwarf virus (CLRDV), and other virus-specific sequence contigs, were de novo assembled according to Ramos-Sobrinho et al. (2021). To further investigate the presence of CLRDV in cacao leaves, total RNA was individually extracted using a modified silica protocol (Rott and Jelkmann, 2001) and used as template for cDNA synthesis with random hexamers using the SuperScript™ IV First-Strand Synthesis System (Invitrogen, CA, USA) following the manufacturer´s protocol. Detection of CLRDV was carried out by reverse transcription-polymerase chain reaction (RT-PCR) with the primers PL4F and o3-R, which amplify the open reading frame 3 (ORF3) encoding the capsid protein (Corrêa et al., 2005). Expected size amplicons (~0.6 kb) were observed from 16 out of 30 symptomatic plants, indicating ~53% of the cacao trees were infected by CLRDV. Considering 14 symptomatic plants tested negative for CLRDV, the symptoms observed here could also be caused by other viral groups or abiotic stress. To confirm the detection of CLRDV, the first half (~3.5kb) of the viral genome was amplified from two representative cacao samples using the primers P20F and P22R (Avelar et al., 2020). The RT-PCR products were gel-purified using the Wizard® SV Gel and PCR Clean-Up System (Promega, WI, USA) and Sanger sequenced. The RNA Illumina sequencing from pooled cacao samples (n=30) yielded 34,610,572 million trimmed reads. Two contigs of 868 and 839 nucleotides (nt) in length, and sharing high nt identity with CLRDV isolates, were assembled from 6,903 and 10,271 reads, at a coverage depth of 795 and 1,224x, respectively. Together, these contigs represent ~29% of the complete viral genome and included part of the 5´-untraslated region, ORF0 and the second half of ORF1-ORF2. Additional CLRDV-like contigs were observed across the viral genome, but they were not considered for further analyses due to the poor sequence quality. The Illumina- and Sanger-derived ORF0 and partial ORF1-ORF2 sequences shared >97% nt identity, suggesting they were congruent. Pairwise sequence comparisons for ORF0, encoding the gene silencing suppressor P0, indicated the cacao-associated isolates shared 99.7 and 99.2% nt and amino acid (aa) identity one with another, respectively. The ORF0 nt sequences showed 91.9-93.8 and 90.7-93.6% identity, while the aa sequences shared 85.8-88.5 and 86.2-90.0% similarity, with CLRDV isolates previously reported in South America and the USA, respectively. Finally, the ~3.5kb nt sequences of cacao-infecting CLRDV isolates shared 92.9-95.8% identity with CLRDV genomes deposited in NCBI-GenBank. The Bayesian phylogenetic tree reconstructed based on ORF0 nt sequences showed the new sequences were more closely related to CLRDV-atypical isolates (GenBank accession nos. KF359946, KF359947, KF906260, and KF906261). Together, these results suggest the new ORF0 sequences belong to CLRDV and were deposited in GenBank under accession nos. ON954058-ON954059. To our knowledge, this is the first report of CLRDV infecting cacao plants, expanding the range of malvaceous hosts of this polerovirus. CLRDV is largely known for causing yield losses in cotton crops, but additional studies are needed to determine if CLRDV infection is detrimental to cacao production.

Case Report: Asymmetric Changes of Ophthalmic Findings in Bilateral Solar Eclipse Maculopathy.

A more profound understanding of the relationship between ophthalmic findings and eye dominance is needed to guide the timely and proper treatment of binocular photic maculopathy or other macular degeneration. A patient with binocular solar maculopathy presented with asymmetric ophthalmic manifestations and changes over 14 months of observation. Early recognition of solar maculopathy can be vital for early diagnosis and better prognosis. A 21-year-old male was reported to have blurred vision after staring directly at an annular solar eclipse several times for a few seconds on June 21, 2020. His Snellen best-corrected visual acuity (BCVA) declined to 20/50 in both eyes. He was right-eye dominant according to the hole-in-the-card test. The fundoscopy found a yellow spot in the center of both maculae. Spectral domain optical coherence tomography (SD-OCT) images revealed a full thickness hyperreflectivity extending from the inner retinal layers to the retinal pigment epithelium (RPE) along with a localized disruption of the ellipsoid portion of the inner segments and RPE in both eyes. The medical history and clinical manifestations described above supported the diagnosis of solar maculopathy. SD-OCT images during follow-up indicated a diminishing disruption of the ellipsoid portion of the inner segments and RPE without treatment. Interestingly, the non-dominant left eye made a better anatomical recovery. Finally, the Snellen BCVA remained 20/20 in both eyes after 14 months. Binocular solar maculopathy can present asymmetrically and recover asymmetrically as well. The dominant eye suffered more obvious damage and poorer anatomical recovery than the non-dominant eye.

First report of Alternaria alternata causing leaf spot of Cynanchum atratum Bunge in China.

Cynanchum atratum Bunge belongs to Asclepiadaceae, and is distributed in North Korea, Japan and China. Its roots and rhizomes have antibacterial, antiviral, anti-inflammatory and anti-tumor effects. In July 2021, a leaf spot was observed in a 1.3 ha plantation of C. atratum in Harbin, Heilongjiang Province in China. The incidence was more than 85%. Initial symptoms were yellowing leaves with circular, or ellipsoid brown spots forming on leaf apexes or leaf margins. Small spots expanded and coalesced to form large circular or irregular, pale to light brown lesions, and leaves finally withered. Thirty, 5 × 5 mm, leaf pieces excised from the junction of symptomatic and healthy tissues were collected from different leaves with typical symptoms on ten plants, sterilized in 75% ethanol for 30s, then in 2% NaClO for 30s, rinsed in sterile water three times, placed on potato dextrose agar (PDA) plates, incubated for 5 days at 28°C in the dark, further purified by single spore method and transferred to new PDA and potato carrot agar (PCA) plates. Finally, 12 fungal isolates, most with similar morphology, were selected. After a 7-day incubation in the dark, colonies on PDA were 53 to 70 mm in diameter, circular and grayish brown. A total of 150 conidia were evaluated for morphology. Conidia were single or in chains, ovoid to inverted pear-shaped, with 2 to 6 transverse septa, 0 to 4 longitudinal or oblique septa, and measured 16.5 to 56.5μm × 9.0 to 16.5 μm. Beaks and supposititious beaks were mostly columnar, rarely conical, 0 to 22.5 μm × 2.5 to 4.0 μm. Conidiophores were solitary or clustered, pale brown, erect or bent, branched or unbranched, separated, 53.5 to 120.5 μm × 2.5 to 6.0 μm (Fig 1). Based on morphological characteristics, the fungus was identified as Alternaria alternata (Simmons 2007). Two representative isolates (BW and BW2) were used for molecular identification. Internal transcribed spacer rDNA regions (ITS), RNA polymerase II second largest subunit (RPB2), Alternaria major allergen (Alt a 1), translation elongation factor 1-alpha (TEF-1 α) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene were amplified and sequenced with the primers ITS1/ITS4 (White, et al. 1990), RPB2-5F2/RPB2-7CR (Khodaei and Arzanlou. 2013), Alt-F /Alt-R (Hong et al. 2005), TEF-F/TEF-R (Carbone and Kohn. 1999) and GDF/GDR (Templeton et al. 1992). The sequences obtained were deposited in GenBank (ITS: OM317915, ON534349; RPB2: OM296253, ON550475; Alt a 1: OM171248, O550474; TEF: OM238096, O550473; GAPDH: OM296217, ON550472). The phylogenetic analysis of maximum likelihood tree by MEGA 7 showed that the two isolates had 98% similarity with A. alternata CBS 916.96 (Fig 2). To test pathogenicity, 40-day-old plants were sprayed with spore suspensions (1×106 spores /mL) from 7-day-old cultures of BW and BW2. Each isolate was inoculated onto 3 leaves on 3 separate plants. Three other plants were sprayed with sterile distilled water as a control. The plants were incubated in the greenhouse (natural light, T: 25℃, H: 50%). After 15 days, the leaves turned yellow and irregular grayish spots appeared. The fungi reisolated from the inoculated leaves shared the same morphological and molecular features as A. alternata, fulfilling Koch's postulates. No fungi were isolated from the control group. This is the first time to report A. alternata causing leaf spot on C. atratum. Leaf spot can reduce the yields of C. atratum and this study provides a basis for the prevention and control of the disease.

First Report of Diaporthe eres and D. unshiuensis causing Leaf Spots on Sapindus mukorossi in China.

Sapindus mukorossi Gaertn., commonly known as soapberry, is widely cultivated as a landscaping tree in Southern China. In June 2019, a foliar disease with an incidence of ∼60% occurred on trees was observed in the soapberry germplasm repository, Jianning, Sanming, Fujian, China. The symptoms initially appeared as irregular small yellow spots, while the center of the lesions became dark brown with time. Fragments (size 3 to 4 mm2) taken from lesion margins were sterilized and cultured based on Wang et al. Two isolates (FJ1 and FJ21) were obtained with the following morphological characteristics on PDA, (1) FJ1: Conidiogenous cells were 9.7 to 25.0 × 1.5 to 2.2 μm (n=20). Alpha conidia were 6.1 to 8.3 × 2.2 to 3.0 μm (n=30), aseptate, hyaline, smooth, ellipsoidal. Beta conidia were 28.3 to 38.2 × 1.3 to 1.7 μm (n=30), hyaline, smooth, curved to hooked. Conidial drops were milky colored; (2) FJ21: Pycnidia were dark brown, 280 to 843 μm (n=30) in diam., globose, or irregular on alfalfa stems. Conidiophores were hyaline, cylindrical, smooth, and slightly tapered to the apex, 17.4 to 35.4 × 1.5 to 2.6 μm (n=20). Conidiogenous cells were 14.7 to 29.7 × 1.4 to 2.6 μm (n=20). Alpha conidia were 5.6 to 7.1 × 2.4 to 3.4 μm (n= 30), hyaline, smooth, ellipsoidal, or clavate, aseptate, biguttulate. Beta conidia not observed. Conidial drops were yellow. The morphological characteristics of FJ1 and FJ21 were similar to those of Diaporthe spp.. DNA of two isolates was extracted, and the internal transcribed spacer region (ITS) and partial sequences of translation elongation factor 1-alpha (TEF1-α), calmodulin (CAL), β-tubulin (TUB), and histone H3 (HIS) genes were amplified with primers ITS1/ITS4, EF1-728F/EF1-986R, CAL228F/CAL737R, βt2a/βt2b, and CYLH3F/H3-1b, respectively. The sequences were deposited in GenBank (accession nos. MW585608 and MW768905 to MW768908 for FJ1; MT755625 and MT776728 to MT776731 for FJ21). The BLASTn results showed that the ITS, TEF1-α, TUB, HIS, and CAL sequences of FJ1 were 100, 99, 98, 98, and 99% identical to those of D. eres (NR144923, KJ210550, KJ420799, KJ420850, and KJ434999, respectively). For FJ21, BLASTing with the same loci showed 100, 100, 100, 99, and 100% similarity with those of D. unshiuensis (MH121530, MH121572, MH121607 MH121488, and MH121448, respectively). Phylogenetic analyses with the concatenated sequences placed FJ1 and FJ21 in the clades of D. eres and D. unshiuensis, respectively. Pathogenicity tests were performed by wounding leaves of 2-year-old soapberry seedlings with a sterile needle. The leaves were inoculated with D. eres and D. unshiuensis isolates, respectively, with 10 µl of conidial suspensions (106 conidia/ml). Three plants were used for each treatment, and the leaves of each plant were inoculated. The control was treated with 10 µl of sterile water. The plants were kept in a greenhouse (RH > 80%, 25 ± 2°C). In 5 days, all inoculated leaves showed lesions similar to the field symptoms. Controls were asymptomatic. Diaporthe eres and D. unshiuensis were reisolated from the diseased leaves. No fungus was isolated from the control. Previously, D. biconispora and D. sapindicola were reported as the causal agents of soapberry, but this is the first report of D. eres and D. unshiuensis causing leaf spots on S. mukorossi in China. These data will help develop effective strategies for managing this disease.

First report of Cladosporium cladosporioides causing leaf blight on Sambucus chinensis in China.

Sambucus chinensis, belonging to the Caprifoliaceae family, is an economically large herb plant that is widely cultivated in southern China for its good ornamental characteristics, edible properties, and medicinal values. In July 2021, symptoms of leaf spot were observed on Sambucus chinensis plants in two fields of Chongqing Medicinal Botanical Garden (29º8'26" N, 107º13'23" E) in Nanchuan city, Chongqing, China. Disease incidence was approximately 35 and 50% for each field. The symptoms were initially yellow or black irregular spots on leaves, and then increased to larger dark brown lesions. Finally, the entire infected leaf was blighted, withering, curl and abscission. Ten blight leaves were randomly sampled from fields. Tissues were cut into small pieces and surface sterilized with 75% ethanol for 30 s and sterilized in 2% sodium hypochlorite for 2 min, rinsed thrice with sterile distilled water, plated on potato dextrose agar (PDA) plates, and incubated at 25°C for 7 days in the dark. Later, 20 isolates were obtained from the infected leaves and had similar characteristics. Three isolates were randomly selected (CQ81, CQ82, CQ83) for the further study. Colonies on PDA were olive-green to brown with a velvety texture. Conidia (n=30) were pale- to olive-brown, smooth to verruculose and produced in long, branched chains which were easily disarticulate, single celled, and elliptical to limoniform, and measured as 2.51~4.29 × 1.63~2.14 μm. Conidiophores were solitary, straight or flexous, often unbranched. The DNA of three isolates were extracted and the internal transcribed spacer (ITS) region and translation elongation factor 1-alpha (TEF1-α) were sequenced using primer pairs ITS1/ITS4 (White et al. 1990) and EF1-728F/EF1-986R (Carbone and Kohn 1999), respectively. The sequences of three isolates were 100% identical, and one representative isolate CQ82 were deposited in GenBank (ON387641, ITS; and ON409522, TEF). BLASTn analysis of these sequences showed 99 to 100% nucleotide identity with the sequences of C. cladosporioides CPC 14705 in Korea (Bensch et al. 2010). Phylogenetic analysis using Neighbor-joining method and concatenated sequences (ITS +TEF1) with MEGA7 placed isolate CQ82 in C. cladosporioides with 99% bootstrap support. On the basis of morphological and molecular characteristics, the isolates were identified as C. cladosporioides (Bensch et al. 2010; Nam et al. 2015). A total of sixteen healthy potted plants of S. chinensis were conducted for the pathogenicity test. Eight plants were selected and one shoot of each plant was randomly used for inoculation. Leaves from the shoot of each plant were brushed with 106 conidia/ml suspension of isolate CQ82. Another 8 plants were performed in the same procedure, inoculated with sterile distilled water as control. All plants were covered with plastic bags for two days and then arranged in a greenhouse with 80% relative humidity at 25°C. The pathogenicity test was repeated thrice. After 15 days inoculation, the similar symptoms were observed on the inoculated leaves, whereas controls remained healthy. The pathogen was reisolated from blight tissue and identified as C. cladosporioides by the methods described above. Although this fungus was previously reported to cause leaf disease on many plants (Meneses et al. 2018; Sun et al. 2017), this is the first report of C. cladosporioides causing leaf blight on S. chinensis in China. This study will establish a foundation for controlling the disease.

Onions Selected for Reduced Symptom Expression of Iris Yellow Spot Have Higher Photosynthetic Rates

Bulb onion (Allium cepa L.) is an economically valuable vegetable crop in the United States. Onion production is threatened by onion thrips, which are the vector for Iris yellow spot virus, which is the causal agent of Iris yellow spot (IYS). New Mexico State University (NMSU) breeding lines 12-236, 12-238, 12-243, and 12-337 have exhibited fewer IYS disease symptoms in the field; however, little is known about the effects of the disease on the photosynthesis rate (Pn). We hypothesized that these NMSU breeding lines would have a higher Pn than IYS-susceptible cultivars Rumba and Stockton Early Yellow. To test this hypothesis, a field study was conducted for 3 years at NMSU, and Pn was measured five times throughout each season at 2-week intervals. During bulb development and maturation, which occurred at 10 and 12 weeks after transplanting, all NMSU breeding lines exhibited a higher Pn when compared with that of an IYS-susceptible cultivar. Pn was highest at the end of the vegetative growth stage and decreased as bulbs approached maturation for all cultivars. Additionally, a high Pn at 10 and 12 weeks after transplanting coincided with high bulb weight at harvest. NMSU breeding lines have increased Pn compared with that of IYS-susceptible cultivars and resulted in larger and more marketable bulbs. These results indicate that maintaining Pn may be related to reduced IYS symptom expression of onion.

Phenolics and Flavonoids Content of Wijaya Kusuma Leaves Fractions using Micro-plate Based Assay

Epiphyllum oxypetalum (DC.) or Wijaya Kusuma (Cactaceae) is an ornamental plant which is knownto contain polyphenol compounds. The purpose to determine the levels of total phenolic and flavonoidcompounds in the fractions of Wijaya Kusuma leaves using microplate-based assay. Each sample wasdetermined phenolic content with Folin-ciocalteu reagent and flavonoid content using AlCl3 on themicroplate. Absorption was measured using a microplate reader. Screening of DPPH radical scavengingactivity was identified using the TLC method. The results showed that the extract and fractions ofthe leaves of Wijaya Kusuma contained phenolic and flavonoid compounds. The ether fraction hadthe highest phenolic content (35,912±0,54776 mgGAE/g) and flavonoids (0,773±0,008 mgQE/g)compared to other fractions (ether>water>ethyl acetate fraction). There is one yellow spot with an Rfvalue of 0,85 in the ether fraction which is thought to be a compound capable of scavenging the DPPHradicals. The ether fraction of Wijaya Kusuma leaves has potential as a source of polyphenols withantioxidant activity. However, further research is needed to evaluate the antioxidant capacity in theether fraction of Wijaya Kusuma leaves.Keywords: Antioxidant, Epiphyllum oxypetalum (DC.) Haw, Flavonoids, Fraction, High-throughputMicroplate Assay, Phenolics.

First report of Curvularia penniseti causing leaf blight of Bajra Napier hybrid grass in India.

Bajra Napier hybrid (Pennisetum glaucum x Pennisetum purpureum) is a perennial, high yielding grass and is widely grown for fodder in India. During August-2021, Bajra Napier hybrid germplasm line (NBN 15-2) showed severe leaf blight symptoms at ICAR-Indian Grassland and fodder research institute, Jhansi (25.527890 N, 78.5451400 E). Symptoms were initial irregular yellow spots on the leaf lamina, which later became brownish, coalesced and gave blighted appearance to the leaf surface. Disease severity recorded was 55 to 60 percent. To isolate the pathogen, 10 symptomatic leaf samples were cut into small pieces (~4 mm2), surface-sterilized with 70% ethanol for 30 seconds and rinsed with sterile water. Sterilized leaf pieces were transferred to potato dextrose agar (PDA) and incubated at 28°C for 7 days. Four similar fungal isolates (BNHCP-1 to BNHCP-4) were obtained from the affected portions. The colonies were grayish-brown with dark brown margins. Conidia were mostly clavate, elongated, straight or bent at the terminal cell, with 2-3 septa with dimensions of 17.5 to 30 μm × 10 to 12.5 μm (avg. 24 μm × 12 μm; n=40). The third cell from the base was broader and darker. These morphological characteristics were consistent with previous descriptions of Curvularia penniseti (Mitra) Boedijn (Ellis, 1971). To confirm the species, BNHCP-1 was chosen as representative isolate for further studies. Internal transcribed spacer (ITS) region and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene of isolate BNHCP-1 was amplified with primers ITS1F/ITS4R (White et al. 1990) and GDF/GDR (Templeton et al. 1992), and sequenced. The sequences were deposited in GenBank (ITS: OM073980; GAPDH: OM103702.2). BLASTn analysis showed 99.6% and 98% similarity of ITS and GAPDH gene respectively with GenBank accession numbers MH859833.1 (548 bp/550 bp) and MN688838.1 (130 bp/133 bp) of C. penniseti. A maximum-likelihood phylogenetic analysis based on concatenated sequences of ITS and GAPDH gene using MEGA X placed the isolate BNHCP-1 within a clade comprising C. penniseti. Pure culture of BNHCP-1 was deposited in National Agriculturally Important Microbial Culture Collection (NAIMCC), Maunath Bhanjan (Uttar Pradesh) with accession number NAIMCC-F-04251. For pathogenicity, root slips of Bajra Napier hybrid germplasm line NBN 15-2 were transplanted in pots (6 pots; 2 root slips per pot) and kept for fresh growth in a growth chamber at 25 0C for 21 days. Bajra-Napier hybrid plants were sprayed until runoff with conidial suspension (5 × 105 conidia/ml) made from 2-week old fungal colony grown on PDA petri dish. The pots were covered with plastic bag for 48 h to maintain humidity. Inoculated plants displayed small, brown, oval-shaped lesions within seven days on the lamina and edges of the leaf which later enlarged and gave blighted appearance to the leaf. Control plants were asymptomatic. The pathogen was re-isolated from the inoculated leaves and confirmed morphologically, fulfilling Koch's postulates. C. penniseti has been reported earlier from Pennisetum americanum, P. clandestinum, Sorghum and Triticum sp. from different parts of the world (Sivanesan, 1987). However, there is no report of C. penniseti in Bajra Napier hybrid. Thus, to the best of our knowledge, this is the first report of C. penniseti from Bajra-Napier hybrid grass in India. Further studies on economic impact of this disease on Bajra-Napier hybrid production and its presence on commercial cultivars are needed.

3′ UTR is critical for viral RNA accumulation of jasmine virus H

Jasmine virus H (JaVH) is a newly reported viral pathogen of jasmine in China and USA. To study the viral gene function and pathogenic mechanism, a full-length infectious clone of JaVH (pXT-JaVHFJ) was constructed under the control of the cauliflower mosaic virus 35S promoter. pXT-JaVHFJ induced a systemic infection in Nicotiana benthamiana plants by Agro-infiltration, which demonstrated that pXT-JaVHFJ was biologically active. Jasmine showed yellow spots after rubbing with total RNA extracted from Agro-infiltrated N. benthamiana, indicating that JaVH was highly associated with yellow mosaic symptoms observed on jasmine. To investigate the occurrence and mutations of the virus, jasmine samples were collected from eight provinces of China and were tested for JaVH. The samples that were tested positive for JaVH were used to determine the complete genome sequences. They were comprised of 3867 or 3868 nucleotides and their genome organizations resembled that we previous reported for JaVH-FJ. Phylogenetic analyses and sequence comparisons suggest that the eight virus isolates were close isolates of JaVH-FJ and the isolate from Jilin Province was most closely related to JaVH-FJ with 99.2% nucleotide identity over the entire genome and 99.7% identity of coat protein. Further comparative analyses of JaVH-FJ and JaVH-JL revealed additional nucleotide differences in the 3′-untranslated region (3′ UTR). An infectious clone of JaVH-JL and chimeric mutants containing JaVH-FJ or JaVH-JL 3′ UTRs were then constructed for further study. The differential accumulation of JaVH with distinct 3′ UTR suggested that the 3′ UTR of JaVH plays a crucial role in viral RNA accumulation.

First Report of leaf spot disease caused by Enterobacter mori on Canna indica in China.

Canna indica L. is a popular landscape ornamental herb of family Cannaceae throughout China. This plant has been extensively cultivated for decoration and as an ornamental in China. C. indica was seriously affected by a disease in the garden in spring of 2021 with an incidence of 21.2 to 45.6%, and it caused economic loss to control plant diseases with chemicals. Both young and older leaves developed necrotic lesions with small water-soaked spots on leaves, which then enlarged and were bordered by chlorosis. Symptomatic tissues were collected from Zhanjiang city (21.2N110.3E) and Wuchuan city (21.4N110.7E) in Guangdong province. Three symptomatic leaves from each city were surface disinfected in a 1% hypochlorite solution for 3 mins followed by being rinsed in sterile distilled water for 3 times. Six bacterial isolates originated from six single colonies were recovered from the samples. Colonies were raised and opaque with smooth margins. The bacteria were gram-negative, rod-shaped with sizes ranged from 0.4-0.7 μm wide and 1.2-2.0 μm long, without endospore, and were facultative anaerobes. For molecular identification, the direct colony PCR method (Lu et al. 2012) was used to amplify the 16S rDNA (Moreno et al., 2002), gyrB, leuS and rpoB of 2 selected strains, EM21ZJ1 and EM21WC1 using the primer pairs 27F/1492R, gyrB3/gyrB4, leuS3 /leuS4 and rpoBjt112/ rpoBjt748 respectively (Deletoile et al., 2009). The resulting sequences were deposited in GenBank (ON600470 and ON600471 for 16S rDNA; ON600472 and ON600473 for gyrB; ON600474 and ON600475 for LeuS; ON600476 and ON600477 for rpoB). BLAST searches with the four gene sequences revealed the greatest similarity with the sequences of Enterobacter mori (Zhu et al. 2011). The available complete genome sequences of Enterobacter species, especially type strains, were downloaded and the sequences of the corresponding 4 genes of each genome was extracted by Bioedit software. The concatenated sequences were aligned by Mega 11.0 with the neighbor-joining method. Multilocus sequence typing analysis showed that the concatenated sequences of the 2 isolates were clustered with E. mori with 100% bootstrap value. The 2 isolates were selected for pathogenicity tests to fulfill Koch's postulates. Six plants at three- to five-leaf stage were inoculated with each isolate separately, 2 sites of each leaf were inoculated, one site was wounded with a sterile needle and the other was not. The 2 sites of each leaf were covered with a piece of cotton drenched with 200 µl bacteria suspension (108 CFU/ml) from 2 isolates separately. Control plants were inoculated identically except Luria-Bertani (LB) medium was used to drench the cotton. Inoculated plants were placed in an incubator at 25°C, and 80% humidity under a 12-h light/dark cycle for 7 days. After 3 days of incubation, water-soaked yellow spots were observed in all the inoculated plants in both wounded and unwounded sites, except the negative control. The water-soaked yellow spots enlarged and became necrotic that matched those seen in garden. The pathogenicity tests were conducted three times with similar results. The bacteria were then reisolated from the lesions and found to have the same colony morphology and 99.9% identity of 16S rDNA sequences as those of the inoculum. According to morphological and sequence analysis, the pathogen was identified as E. mori. To our knowledge, this is the first report of disease of C. indica caused by E. mori.

Effectiveness of fungicidal protection of spring wheat against powdery mildew and tan spot

The results of the study of the effectiveness of plant protection products against powdery mildew (Blumeria graminis) and tan spot (Pyrenophora tritici-repentis) for spring wheat are presented. It has been noted that the probability of mass destruction of crops by phytopathogens is determined by the stock of the infectious beginning of pathogens, the susceptibility of the host plant and the favorable weather conditions for their development and spread. The experiment was carried out using the methods generally accepted in the Russian Federation. During the observation period, the degree of plant damage of more than 20% in the heading phase was noted in 46% of the years of observation (in 2009, 2013, 2014, 2016, 2017). Epiphytotic development of powdery mildew together with brown leaf rust (Puccinia triticina Eriks.) was registered in 2013–2014 and 2016–2017. For 2009–2019 the degree of plant damage of more than 20% in the heading phase was noted in 46% of the years of observation (in 2009, 2013, 2014, 2016, 2017). Effective protection of wheat against powdery mildew (75–87%) was provided by fungicides based on 2–3 active ingredients. The biological effectiveness of the biofungicide (Bacillus subtilis) was manifested only in the years with moderate wheat damage and did not exceed the average level of 58%. It is more expedient to carry out operational control of yellow spotting of wheat leaves with preparations based on such active substances as "azoxystrobin + epoxiconazole", "tebuconazole + propiconazole". The level of statistically significant yield preserved due to fungicidal protection during the years of depression was 5–6%, with a moderate development of leaf diseases due to chemical fungicides - 18%, and the biological product provided an increase in productivity of 9%. During the years of epiphytoties, the chemical protection of crops retained an average of 24% of the wheat yield, polycomponent preparations had the best performance and stability of action, and the biological protection of plants under these conditions was ineffective.

Leaf spot on Rosa banksiae caused by Calonectriafujianensis in China

Rosabanksiae is a vine-like shrub rose that is endemic to China. In recent years, leaf spot disease hasbeen observed frequently in park in Nanjing City, Jiangsu Province, China. The initial symptoms were small yellow spots; as the disease progressed, the lesions became dark brown. Isolates NLXH2-1-1, NLXH2-2-3, and NLXH 2-4-3 were obtained. Koch's postulates were tested and confirmed that these isolates were pathogenic. Morphologically, macroconidia were cylindrical, rounded at both ends, straight, and (48–) 50–55 (−60) × (2.5–) 3.5–4.5 (−5) μm in length (av. = 52.5 × 4 μm), with 1–3-septate, and gathered conidia bundles. By morphological features and multigene phylogenetic analysis of the six genesITS, act, cmdA, his, LSU and tub2 region, the causal fungus is Calonectria fujianensis.This is the first report leaf spot by Ca. fujianensis on the R. banksiae, provide a theoretical basis for disease prevention and control.

Description of the immature of Podisus graziae Brugnera, Roell & Lemaître, 2021 (Heteroptera: Pentatomidae: Asopinae)

As predaceous insects, the Asopinae have a great economic importance. The knowledge about the development of the species is essential for use in biological control programs. However, only 1% of the Neotropical Asopinae species have the immature stages known. In this work we describe the egg and the five nymphal stages of Podisus graziae Brugnera, Roell & Lemaître, 2021 using light and scanning electron microscopy. The results show that P. graziae eggs and nymphs present unique characters which are not observed in other species of the genus and subfamily. As other Podisus species, the egg present long aero-mycropilar processes, however, the chorion and operculum have a ring of minute spine-like projections. As main diagnostic characters of nymphs, the iridescent coloration of head, thorax and abdominal plates, and a pair of yellow spots in the pronotum and anterior region of abdomen are noticed.

An overview of the most threating diseases that affect worldwide citriculture: Main features, diagnose, and current control strategies

Citriculture has been one of the most important agricultural activities worldwide. Brazil is among the five major citrus producers in the world, together with China, the European Union (EU), the United States, and Mexico. Together, these five groups are responsible for about 50% of the global citrus fruit production and this industrial segment is responsible for generating direct and indirect jobs. However, several citric diseases such as citrus canker, huanglongbing, citrus variegated chlorosis, and citrus black spot have been associated with annual losses of tons of fruits and orange trees impacting the global economy for decades. Citrus canker is caused by the Gram-negative bacteria Xanthomonas citri subsp. citri. and is associated with symptoms such as the formation of chlorotic rings in the leaves, stems, and fruits. Huanglongbing (HLB) is mainly associated with the Gram-negative bacteria Candidatus Liberibacter spp. and its main symptoms are the appearance of yellowish branches and deformed small leaves with yellowish spots. Citrus variegated chlorosis (CVC) is associated with the bacterium Xylella fastidiosa and causes chlorotic spots with irregular edges on leaves and deformation of new leaves. Citrus black spot (CBS) is caused by the fungus Phyllosticta citricarpa and generates lesions on fruits and reddish-brown leaf lesions. Since citrus is important for global agriculture, the current review addresses the main features of these important diseases including their symptoms and transmission, as well as the diagnosis and control strategies that have been studied so far for application in the field.

First report of tomato yellow mottle-associated virus infecting Solanum nigrum in China.

First report of tomato yellow mottle-associated virus infecting Solanum nigrum in China Zhenggang Li, Yafei Tang, Xiaoman She, Guobing Lan, Lin Yu, and Zifu He† Guangdong Provincial Key Laboratory of High Technology for Plant Protection, Plant Protection Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou, 510640, P. R. China. Tomato yellow mottle-associated virus (TYMaV) is a newly found cytorhabdovirus associated with epinasty of leaflet blades, yellow spots, puckering, and mottling symptoms in tomato plants in China (Xu et al., 2017). In May 2020, Solanum nigrum plants exhibiting leaf crinkling and mosaic symptoms (eXtra S1) were found in Shantou city, Guangdong, China. To identify the causal pathogens, the leaves of three symptomatic plants were collected and subjected to total RNA extraction with TRIzol Reagent (Takara, Kusatsu, Japan). About 100 μg RNA mixture, which consisted of an equal amount of total RNA extracted from the three samples, was subjected to small RNA deep sequencing and assembly (sRSA) (Kreuze et al., 2009). Small RNA cDNA library was constructed with the method described previously (Mi et al., 2008). Small RNA deep sequencing was performed with Illumina HiSeq X Ten platform. VirusDetect (Zheng et al., 2017) was used to analyze the sequence data. The result showed that the sequence data includes about 11 million reads and generated 194 unique contigs after removal of host-derived contigs. Subsequently, the unique contigs were screened using BLASTn search against the virus database. One hundred and five unique contigs were mapped to TYMaV genome (reference sequence, KY075646), 21 unique contigs were mapped to RNA1 segment of tomato chlorosis virus (ToCV) genome (reference sequence, KY618796), 67 unique contigs were mapped to RNA2 segment of ToCV genome (reference sequence, KY618797), and one unique contig was mapped to pepper veinal mottle virus (PVMV) genome (reference sequence, FJ617225) (eXtra S1). To verify the sRSA result for TYMaV detection, RT-PCR was performed with two primer pairs TYMaV-F1/R1 (5'-TCATTAGACTCAGGCCTAATCCTCA AAGT-3'/5'-GATATGGAGACGTCCAAGTTCAAAGGGATGGA-3'), and TYMaV-F2/R2 (5'-TATGCGGCAGCTTTCATGTCTATAGACCCT-3'/5'-ATGACCTAGCTTCAATAACAGTCGCG-3'), which are designed according to the sRSA result. All the symptomatic samples tested positive for TYMaV (eXtra S2). Western blot with TYMaV N protein-specific antibody further verified the result (eXtra S2). To obtain the nearly full-length sequence of TYMaV identified in Shantou, 13 primer pairs were designed to amplify the viral fragments. The amplified PCR products were then introduced into pMD19T (Takara, Kusatsu, Japan) and sequenced by Sangon Biotech Co. (Shanghai, China). The nearly full-length sequence of TYMaV Shantou isolate (TYMaV-ST) was assembled from the 13 overlapping sequences (reference sequence, MW527091). TYMaV-ST genome comprises of 13401 nt and shares 84.93% nucleotide sequence identity with the reference genome (KY075646). In addition, 37 S. nigrum samples and 20 tomato samples nearby with viral disease symptoms were collected from different sites of Guangdong province, China. Six S. nigrum samples and five tomato plant samples tested positive for TYMaV by RT-PCR, suggesting a wide spread of the virus in the surveyed region. These results together with those of the sRSA assay also suggest that the disease symptoms shown in the original S. nigrum plants may not necessarily be caused by TYMaV or by TYMaV alone. To our knowledge, this is the first report of TYMaV infecting S. nigrum in China. S. nigrum is a common weed which belongs to the family Solanaceae and may serve as a reservoir for TYMaV in the fields. Further research is needed to verify whether this is indeed the case, and to understand the characteristics of this virus including its transmission, pathogenicity, and economic significance. The authors declare no conflict of interest. Funding This work was supported by the Key Research and Development Program of Guangdong Province (2018B020202006), the Agricultural Competitive Industry Discipline Team Building Project of Guangdong Academy of Agricultural Sciences (202103TD and 202105TD), the Science and Technology Program of Guangzhou (202102020504), and Special Fund for Scientific Innovation Strategy-Construction of High-Level Academy of Agriculture Science (R2019PY-QF003).

First Report of Puccinia modiolae Causing Rust Disease on Malva verticillata in Korea.

Malva verticillata (Malvaceae), commonly called Chinese mallow or whorled mallow, is an annual herb native to East Asia and is currently distributed worldwide. In Korea, this plant is cultivated as a leafy vegetable and cooked like spinach or used in soups and also as a medicine material. In March 2022, typical symptoms of rust disease were observed on M. verticillata in a plastic house (37°22'12″ N, 127°34'30" E) in Yeoju, Korea. Yellow or light green round chlorotic spots appeared on the upper surface of infected leaves, while reddish-brown or dark brown rust pustules formed on its lower surface. Infection occurred in 10% of M. verticillata plants surveyed, and disease severity ranged between 30-90%. A representative sample was deposited in the Kunsan National University Herbarium (KSNUH1762). Telia were mostly hypophyllous, reddish-brown to dark brown, round, mostly grouped, and 0.3-0.7 mm in diameter. Teliospores were mostly two-celled, but rarely one or three-celled, yellowish to light brown, fusoid, and 42.9-101 × 10.8- to 18.8 μm (average 72.7 ± 12.3 × 14.2 ± 1.92 μm [mean ± SD]; n = 50), with a smooth, hyaline to yellowish wall of 1.0-2.5 μm thickness. The morphological characteristics were similar to those reported for Puccinia modiolae (Aime and Abbasi 2018; Albu et al. 2019). To confirm the morphological identification, genomic DNA was extracted from the teliospores of an infected leaf. The internal transcribed spacer (ITS) with primers ITS5-u and ITS4rust (Pfunder and Schürch 2001) and the large subunit (LSU) rDNA with primers LRust1R and LRust3 (Beenken et al. 2012) were amplified for sequencing. The resulting sequences were deposited in GenBank with accession numbers ON631218 for ITS and ON631226 for LSU. BLASTn search showed that the Korean sample was identical to the ITS sequences of P. modiolae from Modiola caroliniana (MK458693-MK458697) and the LSU sequences from M. caroliniana, Malva sylvestris, and Alcea rosea (MH742976, MH742977, and MH742978). In the phylogenetic trees of the ITS and LSU sequences, the Korean sample was grouped with the reference sequences of P. modiolae, with the maximum supporting value. For the pathogenicity test, rust-infected leaf discs were placed on the upper or lower surfaces of leaves of three healthy M. verticillata. Three non-inoculated plants served as controls. Inoculated and non-inoculated plants were maintained in a growth chamber at 22°C, a 16/8 h light cycle, and 80% humidity. After three weeks, all inoculated plants developed evident rust symptoms on the upper and lower surfaces of the leaves on which the leaf discs were placed, whereas the control plants remained symptomless. The pathogen present on the inoculated plants was confirmed to be the same pathogen as that observed in the field, fulfilling Koch's postulates. Based on the morphological investigation, sequence analysis, and pathogenicity tests, P. modiolae was identified as the causal agent of rust disease on M. verticillata. To date, this pathogen has been reported on seven Malvaceae plants, including Alcea rosea, Althaea officinalis, Lavatera arborea, Malva parviflora, Malva sylvestris, Modiola caroliniana, and Modiola sp., in North and South America (Farr and Rossman 2022). However, it has not been reported in Asia or Korea. This study is the first report of rust disease on M. verticillata worldwide. Considering its high incidence rate and severe damage, this pathogen is a potential concern for the cultivation of M. verticillata in Korea. This finding could contribute to developing phytosanitary and control treatments for this disease.