Neighbor-joining Method Research Articles

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.

A DNA barcode reference library for CITES listed Malagasy Dalbergia species.

On Madagascar, the illegal and unsustainable exploitation and illegal international trade of Dalbergia (rosewood) precious woods remain a serious conservation problem. Members of this genus are at high risk of extinction as a consequence of logging, mining, and slash and burn agriculture. Morphological identification of these Malagasy species is difficult in the absence of flowers and fruits, especially in the case of cut trees, sawn wood, and finished product. In this study, we use molecular barcoding to identify the Dalbergia species with the intent to contribute to the control of their illegal trade. Thirty-six Dalbergia samples representing 12 Malagasy species of which 11 have high commercial value, were collected to test the efficacy of a region of the plastid genome (rbcL) and a nuclear-transcribed ITS for barcoding. These widely used markers, as well as DNA barcoding gaps, "best match" and "best close match" approaches, and the neighbor-joining method were employed. All samples were amplified and sequenced using the two markers. Using a single locus, the "best match" and "best close match" approaches revealed that ITS has high discriminatory power within the tested Malagasy species. The combination of rbcL + ITS revealed 100% species discrimination. This study confirms that ITS alone and in combination with chloroplast barcode rbcL allow non-ambiguous identification for the 12 species studied. The results contribute to the development of DNA barcoding as a useful tool to identify Malagasy Dalbergia and suggest that the approach developed should be expanded to all 56 potentially exploited species in reference to international CITES requirements and the sustainable management of valuable resources.

MANGIFERA KEMANGA BLUME (ANACARDIACEAE) TAXONOMIC ASSESSMENT FOR GENETIC DIVERSITY BASED ON MOLECULAR SUBSTANTIATION

Mangifera kemanga Blume is a wild relative of mango (Mangifera indica) and a local fruit with various potential uses. The community uses its fruit and leaves for food and its wood as a building material. However, the genetic diversity and taxonomic status of M. kemanga species still need more exploration and have a dispute with M. caesia Jack. The presented study aimed to determine the taxonomic status of M. kemanga and its relationship with M. caesia through a molecular approach and to recognize the species’ delimitation. The recorded data analysis used the trnL-trnF intergenic spacer sequence. Carrying out DNA isolation employed the Geneaid Genomic DNA Mini Kit (Plant) protocol, with the DNA sequences analyzed for kinship using Maximum Parsimony and Neighbor-Joining methods and genetic diversity analysis performed using DnaSP 6. A total of 27 trnL-trnF intergenic spacer sequence identification and authentication resulted from BLAST on NCBI as sequences derived from the genus Mangifera. The phylogenetic tree revealed that the accessions of M. kemanga and M. caesia are very similar, hence, considered not as independent species. Thus, based on the trnL-trnF intergenic spacer sequences, M. kemanga is a synonym of M. caesia with taxonomic status as a variety of M. caesia. This molecular-based taxonomic evidence is significant for determining the valid species status of M. kemanga so that it can provide basic information for further studies on biodiversity and germplasm conservation.

Isolation, characterization, therapeutic potency, and genomic analysis of a novel bacteriophage vB_KshKPC-M against carbapenemase-producing Klebsiella pneumoniae strains (CRKP) isolated from Ventilator-associated pneumoniae (VAP) infection of COVID-19 patients

BackgroundCarbapenem-resistant Klebsiella pneumoniae (CRKP) is a significant clinical problem, given the lack of therapeutic options. The CRKP strains have emerged as an essential worldwide healthcare issue during the last 10 years. Global expansion of the CRKP has made it a significant public health hazard. We must consider to novel therapeutic techniques. Bacteriophages are potent restorative cases against infections with multiple drug-resistant bacteria. The Phages offer promising prospects for the treatment of CRKP infections.ObjectiveIn this study, a novel K. pneumoniae phage vB_KshKPC-M was isolated, characterized, and sequenced, which was able to infect and lyse Carbapenem-resistant K. pneumoniae host specifically.MethodsOne hundred clinical isolates of K. pneumoniae were collected from patients with COVID-19 associated with ventilator-associated acute pneumonia hospitalized at Shahid Beheshti Hospital, Kashan, Iran, from 2020 to 2021. Initially, all samples were cultured, and bacterial isolates identified by conventional biochemical tests, and then the ureD gene was used by PCR to confirm the isolates. The Antibiotic susceptibility test in the disc diffusion method and Minimum inhibitory concentrations for Colistin was done and interpreted according to guidelines. Phenotypic and molecular methods determined the Carbapenem resistance of isolates. The blaKPC, blaNDM, and blaOXA-23 genes were amplified for this detection. Biofilm determination of CRKP isolates was performed using a quantitative microtiter plate (MTP) method. The phage was isolated from wastewater during the summer season at a specific position from Beheshti Hospital (Kashan, Iran). The sample was processed and purified against the bacterial host, a CRKP strain isolated from a patient suffering from COVID-19 pneumoniae and resistance to Colistin with high potency for biofilm production. This isolate is called Kp100. The separated phages were diluted and titration by the double overlay agar plaque assay. The separate Phage is concentrated with 10% PEG and stored at −80 °C until use. The phage host range was identified by the spot test method. The purified phage morphology was determined using a transmission electron microscope. The phage stability tests (pH and temperature) were analyzed. The effect of cationic ions on phage adsorption was evaluated. The optimal titer of bacteriophage was determined to reduce the concentration of the CRKP strain. One-step growth assays were performed to identify the purified phage burst’s latent cycle and size. The SDS-PAGE was used for phage proteins analysis. Phage DNA was extracted by chloroform technique, and the whole genome of lytic phage was sequenced using Illumina HiSeq technology (Illumina, San Diego, CA). For quality assurance and preprocessing, such as trimming, Geneious Prime 2021.2.2 and Spades 3.9.0. The whole genome sequence of the lytic phage is linked to the GenBank database accession number. RASTtk—v1.073 was used to predict and annotate the ORFs. Prediction of ORF was performed using PHASTER software. ResFinder is used to assess the presence of antimicrobial resistance and virulence genes in the genome. The tRNAs can-SE v2.0.6 is used to determine the presence of tRNA in the genome. Linear genome comparisons of phages and visualization of coding regions were performed using Easyfig 2.2.3 and Mauve 2.4.0. Phage lifestyles were predicted using the program PHACTS. Phylogenetic analysis and amino acid sequences of phage core proteins, such as the major capsid protein. Phylogenies were reconstructed using the Neighbor-Joining method with 1000 bootstrap repeat. HHpred software was used to predict depolymerase. In this study, GraphPad Prism version 9.1 was used for the statistical analysis. Student’s t-test was used to compare the sets and the control sets, and the significance level was set at P ≤ 0.05.ResultsPhage vB_KshKPC-M is assigned to the Siphoviridae, order Caudovirales. It was identified as a linear double-stranded DNA phage of 54,378 bp with 50.08% G + C content, had a relatively broad host range (97.7%), a short latency of 20 min, and a high burst size of 260 PFU/cell, and was maintained stable at different pH (3–11) and temperature (45–65 °C). The vB_KshKPC-M genome contains 91 open-reading frames. No tRNA, antibiotic resistance, toxin, virulence-related genes, or lysogen-forming gene clusters were detected in the phage genome.Comparative genomic analysis revealed that phage vB_KshKPC-M has sequence similarity to the Klebsiella phages, phage 13 (NC_049844.1), phage Sushi (NC_028774.1), phage vB_KpnD_PeteCarol (OL539448.1) and phage PWKp14 (MZ634345.1).ConclusionThe broad host range and antibacterial activity make it a promising candidate for future phage therapy applications. The isolated phage was able to lyse most of the antibiotic-resistant clinical isolates. Therefore, this phage can be used alone or as a phage mixture in future studies to control and inhibit respiratory infections caused by these bacteria, especially in treating respiratory infections caused by resistant strains in sick patients.

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.

First Report of Leaf Spot on water convolvulus Caused by Stemphylium solani in Yunnan Province, China.

Water convolvulus (Ipomoea aquatica Forsk.), a member of the Convolvulaceae family, is an important tropical vegetable cultivated in China (Liu et al. 2017). From 2016 to 2020, dark-brown leaf spots were observed in major water convolvulus (cv. Large leaf) growing areas (2 ha) in Honghe City (24°12' N, 103°6' E), Yunnan Province, China. Field investigations showed that a leaf spot disease occurred on water convolvulus in four fields with 15% incidence (50 plants in each field were investigated) and resulted in up to a 10% decrease in its total production. Symptoms on water convolvulus plants appeared as small lesions, yellowish-green and circular on the leaves. Ten plants were selected randomly from the growing area, with three diseased leaves collected from each plant. Symptomatic tissues were excised, surface sterilized with 75% ethanol for 30 s, washed in sterile-distilled water three times, and placed on the Potato Dextrose Agar (PDA) followed by incubation at 25°C in the dark for 7 days. Colonies on PDA were gray to green in color and fuzzy in the middle, with irregular borders. Conidiophore morphology showed single, yellowish-brown or brown structures with 1~6 septa, and long 22~125 µm, wide 3.5~5.5 µm. Conidia were elliptical, black-brow, solitary, with a smooth surface, 1~6 longitudinal septa and 1~3 transverse septa, 20~30 µm in length, and 15~22 µm in width. The morphological characteristics of the fungus were consistent with the description of Stemphylium solani (Chai et al. 2014; Weber, 1930). To further confirm the identity of the 30 isolates, the partial gapdh (glyceraldehyde-3-phosphate dehydrogenase), tef1 (translation elongation factor 1-alpha), cmdA (Calmodulin) and ITS (intemal transcribed spacers) sequences were amplified by PCR with the primer pairs of gpd1/gpd2, EF1-728F/EF1-986R, CALDF1/CALDR2 and ITS1/ITS4, respectively (Berbee et al. 1999; Carbone & Kohn. 1999; Lawrence et al. 2013; White et al. 1990). Multiple sequence alignments were generated using MEGA7, and phylogenetic analysis was conducted with the neighbor-joining (NJ) method (Tamura et al. 2007), the results indicated that all sequences from the 30 isolates were identical. Thus, one representative isolate, KXC11033003 was chosen for further analysis. The ITS, gapdh, cmdA and tef1 sequences of this isolate were submitted to the NCBI GenBank database (accession nos. OL444947~OL444950). The strain KXC11033003 and S. solani (CBS-408.54) formed a clade with 82% bootstrap value (Figure S2). To fulfill Koch's postulates, 30 plants were inoculated for each of the thirty isolates. Conidia were sprayed on leaves of water convolvulus (8-true-leaf stage) in a suspension of 107 conidia/mL or water as a healthy control in a greenhouse at 15~18℃ (night) / 25~28℃ (day) with 95% humidity. Symptoms of dark brown spots appeared on the leaves after 7 days, whereas controls remained healthy The pathogens were reisolated from the lesions and confirmed identical to the original isolate by gene sequences. No pathogens were isolated from the control plants. To our knowledge, this is the first report of leaf spot caused by S. solani on water convolvulus in Yunnan Province, China. Further, Stemphylium leaf spot caused by S. solani has been reported previously on tomato, garlic, pepper (Zheng et al.2008; Nasehi et al.2018). This study stresses the need to identify appropriate management strategies for S. solani that help prevent quality and yield losses in water convolvulus in China.

Cloning, Characterization, and Expression Prolife Analysis of Tyrosinase Genes: Insights into Black Shell Formation in Cyclina sinensis

Shell colors that exhibit a positive relationship with excellent traits can be employed as marker colors for breeding new varieties of bivalves. The clam Cyclina sinensis is an economically important marine bivalve that has three main colors: black, white, and purple. In the present study, we cloned and analyzed the full-length cDNA of the tyrosinase gene (TYR), which is the key gene for melanin synthesis, to explore the formation mechanism of black shells. The full-length cDNA of TYR was cloned by RACE-PCR, and the results showed that the full-length cDNA of TYR was 2993 bp, including a 2262 bp open reading frame encoding 753 amino acids. The results of functional domain prediction showed that deduced TYR protein contains a typical functional domain of tyrosinase, the common central domain. Phylogenetic analysis was conducted using neighbor-joining and maximum likelihood methods, and the results indicated that the evolutionary positions of C. sinensis and Meretrix meretrix were closest. The qRT‒PCR results showed that TYR mRNA was highly expressed in the outer edge of the mantle, which suggested that TYR was involved in the synthesis of melanin in the mantle of C. sinensis and might play an important regulatory role in the formation of melanin in black clam shells.

First Report of Enterobacter roggenkampii and E. cloacae complex sp. Caused Tomato Pith Necrosis in Guangdong Province, China.

Tomato (Solanum lycopersicum L.) is an important greenhouse and field-grown vegetable. During 2019 to 2021, a new bacterial pith necrosis broke out in tomato producing areas in China. The disease incidence rate in the field was approximately 10% to 30% in a few tomato planting areas of Guangdong province, and even 100% in Dianbai distinct, Maoming city. Diseased plants showed yellowing of the lower leaves, brown vascular tissues, and wilting along with brown necrotic spots and a large number of adventitious roots on the stem. Diseased plants were collected, and short fragments of the diseased stems were sterilized with 75% alcohol for 2 minutes, washed with sterile water twice, and stripped the cortex (Fang 1998). Dilutions of xylem specimen soaking solution were plated onto the TTC medium (peptone 10.0 g, acid hydrolyzed casein 1.0 g, glucose 5.0 g, agar 15.0 g, distilled water 1000 mL, 0.5% 2, 3, 5-triphenyltetrazolium chloride, pH7.0), and cultured at 28℃ for 24 h. Three pink single colonies (A2 from Guangzhou (113°21' E, 23°9' N), Guangdong, and K6, and K7 from Maoming (110°55' E, 21°25' N), Guangdong) were selected and purified. Strains A2, K6, and K7 were Gram-negative, motile, and showed white fluidal colonies with pink center on TTC medium, and white, round, and smooth-surface colonies on NA medium (peptone 10 g, beef extract 3 g, sodium chloride 5 g, agar 15 g, distilled water 1000 mL, pH7.0) at 28℃ for 24 h. Three strains could utilize citrate, sorbitol, lactose and arginine, and were negative for methylred reaction test, determination of phenylalanine amino acid deaminase, lysine decarboxylase, urease, soluble starch decomposition and gelatin liquefaction, whereas were positive for Voges-Proskauer test, which conformed to the characteristics of genus Enterobacter (Davin-Regli et al. 2019). To determine the species of the Enterobacter isolates, partial sequences 16S rDNA, gyrB, and rpoB of strain A2, K6, and K7 were amplified. The PCR products were purified, sequenced, and deposited to GenBank. The BLASTN analysis of 16S rDNA, rpoB and gyrB sequences showed strain A2 (MW785888, OL364948, OL364943) was 99.20%, 99.17% and 98.57% identity with E. roggenkampii DSM16690, respectively, strain K6 (MW785890, OL364950, OL364945) was 99.73%, 99.63%, 99.63% identity with E. cloacae complex sp. N13-01531, and strain K7 (MW785893, OL364951, OL364946) was 99.8%, 98.81%, 98.99% identity with the E. roggenkampii Ed-982 and Ek140. Nucleotide sequences of 3 strains were aligned using ClustalW program, and neighbor-joining method (NJ) was used in the construction of a phylogenetic tree using MEGA7 program. Phylogenetic trees based on gyrB sequence, rpoB sequence, and the concatenated sequence of 16S rDNA-rpoB-gyrB and rpoB-gyrB showed strain A2 and K7 were clustered to E. roggenkampii, strain K6 was clustered to E. cloacae complex sp. The roots of tomato material 'Moneymaker' at stage of 4-5 true leaves were cut and irrigated 10 mL bacterial suspension (OD600=0.6) of strains A2, K6, and K7, respectively. As a control, the tomato roots were treated with 10 mL sterile water. All plants were incubated at 30°C. The experiments were conducted with 20 tomato seedlings for each tested strain and control, and repeated twice. All plants inoculated showed yellowing in the lower leaves 6-7 days after inoculation (DAI), subsequently the stems of some plants were rotten, along with bacterial pus in the internodes. The plants wilted, and stems were hollow 20 DAI, which is similar to the field symptoms. No symptoms were observed in control plants. Strains were successfully reisolated from wilting plants, and identified as A2, K6, and K7, respectively, based on gyrB sequence analysis, fulfilling Koch's postulates. Zhou et al. (2021) reported that E. roggenkampii caused bacterial wilt of mulberry in Guangxi, China. Chen et al. (2021) reported E. asburiae caused tomatoes pith necrosis in Fujian and Zhejiang, China. To our knowledge, this is the first report of E. roggenkampii and E. cloacae complex sp. causing bacterial pith necrosis of tomato. Further research would focus on exploring the pathogenic mechanism of the pathogen, and providing reference of controlling the disease.

First Report of Rhizopus arrhizus (syn. R. oryzae) Causing Garlic Bulb Soft Rot in Hebei Province, China.

Rhizopus soft rot occurs on the succulent tissues of vegetables, fruits, and ornamental plants throughout the world (Cui et al. 2019). When the garlic is in the seedling stage in the fields (Fig. S1) in November 2021, a disease outbreak on garlic bulbs suspected as Rhizopus soft rot occurred in Daming County, Handan City, Hebei Province of China (N 36°17', E 115° 13'). This disease symptom was first found in the garlic seedling stage in China. Disease incidence was 10% to 30% in cultivated garlic bulbs. There were soft water-soaked lesions on the surface of diseased garlic bulbs and the interiors were brown and soft. In the disease severe field, white to gray mycelia were observed on the diseased garlic bulbs. Infected garlic bulbs were sampled to isolate and determine the identity of the disease-causing organism. Symptomatic bulbs were surface sterilized with 1% NaClO for 2 min, dipped in 75% ethanol for 3 min and rinsed three times with autoclaved distilled water. Small pieces of the inner decayed tissue were removed and cultured on potato dextrose agar (PDA) at 28°C for 2 to 3 days. Five white colonies grew on PDA and then they became brownish gray to blackish-gray mycelium. The fungal strains were purified by hyphal-tip isolation method. To determine the identity of the five isolated fungi, we analyzed their internal transcribed spacer (ITS) region sequences (Jung et al. 2012). BLAST analysis of the ITS sequences from DSF-0-2 (accession no. ON706022), DSF-0-3 (accession no. ON706021), DSF-0-4 (accession no. ON706020), DSF-0-5 (accession no. ON706019) and DSF-0-6 (accession no. ON706018) were all 100% identical with Rhizopus arrhizus (syn. Rhizopus oryzae). Phylogenetic trees were constructed using the neighbor-joining method of MEGA11 based on the sequences of ITS rRNA gene (Walther et al. 2013). Phylogenetic trees indicated that isolates were most likely Rhizopus arrhizus (syn. Rhizopus oryzae) (Fig. S2). We selected one isolated strain, DSF-0-2, for characterize the morphology and test its ability to cause garlic bulb soft rot. Under the microscope, nonseptate rhizoids, sporangia, and sporangiospores were observed (Fig. S1). Sporangiospores were unequal, subglobose, numerous irregular, or oval, and 9.7 (6.2 - 12.5) × 6.5 (4.1 - 8.5) μm (n = 50) in diameter. The sporangia were globose, black, 121.5 (65 - 198) μm (n = 50) in diameter. Based on the rDNA-ITS sequencing and the morphological characteristics, the DSF-0-2 isolate was identified as Rhizopus arrhizus (syn. Rhizopus oryzae) (Zheng et al. 2007; Abeywickrama et al. 2020). To complete Koch's postulates, surface-sterilized healthy garlic bulbs were inoculated with R. arrhizus isolate DSF-0-2. A 1.0-ml sterile syringe was used to inject 50 μl of a 106 conidia/ml suspension into each of five healthy bulbs. As a control, garlic bulbs were treated with sterile distilled water. The inoculated and control bulbs were incubated at 28°C for 7 days. The bulbs inoculated with R. arrhizus DSF-0-2 showed symptoms of water soaking, and the tissues were brown and soft throughout the bulb at 7 days (Fig. S1). Results of the three trials were the same. No symptoms were observed in the control group. R. arrhizus was reisolated from the symptomatic garlic bulb and confirmed as such based-on colony and sporangia morphology and ITS sequence. There were some reports that R. arrhizus infects cassava tubers and potato tubers (Amadioha and Markson 2007; Cui et al. 2019). To our knowledge, this is the first report of R. arrhizus (syn. Rhizopus oryzae) associated with soft rot on garlic bulb in the seedling stage in China. This disease has posed a potential threat during garlic seedling stage in the field. Management measures should be considered before this disease outbreaks widely. Garlic bulbs died in the seedling stage, which caused production reduction, serious economic loss and soil pollution. This finding may help to take effective control measures for this disease.

First report of Bougainvillea spectabilis Willd bacterial leaf spot caused by Pantoea stewartii subspecies indologenes in China.

Bougainvillea spectabilis Willd. is an important ornamental flowering plant belonging to the family Nyctaginaceae. It is widely used in landscape designs in tropical and subtropical regions. In December 2020, severe disease-causing leaf spots were discovered on the leaves of B. spectabilis in the Modern Agricultural Park (110°19' E, 21°26' N) Zhanjiang City, Guangdong Province, China. Field surveys revealed that the disease was widespread, with an incidence of 60-80%. Early symptoms on the leaves appeared as tiny leaf spots that later developed into concentric circles surrounded by a yellowish halo (Fig. 1). Diseased leaves with typical symptoms were collected for pathogen isolation. The leading edges of the lesions were excised, sanitized in 75% ethanol for 30 s and in 3% sodium hypochlorite for 3 min, and rinsed three times with sterile distilled water (SDW). The diseased tissue was crushed in 1 mL SDW, soaked for 15 min, and then spread onto nutrient agar medium on a petri dish. Circular, bright yellow colonies with smooth margins were observed after 24 h of incubation at 28 °C. The isolate (SJM1) was a gram-negative bacillus with positive results for catalase, indole synthesis, maltose, and arbutin and negative results for sorbitol, lactose, salicin, and starch hydrolysis. The SJM1 genomic DNA was extracted using the TIANamp Bacterial DNA Kit, and partial 16S rDNA gene segments were amplified using the bacterial generic primers 27F and 1492R. The collated 16S rDNA gene sequences were submitted to the NCBI GenBank (MZ723935). BLAST analysis of the sequences revealed 99.38% identity with Pantoea stewartii (MG517424.1). Amplification using subspecies-specific primers galE (#562/564; Gehring et al. 2014), glmS (#356/341; Wensing et al. 2010), and pstC + pstS (#338/339; Wensing et al. 2010) revealed that the genes showed 99-100% identity with P. stewartii subsp. indologenes (galE = 100%, MZ754494.1; glmS = 99.79%, MZ75496.1; and pstC + pstS = 99.89%, MZ754495.1). Phylogenetic trees were constructed using the neighbor-joining method (MEGA X), with both the 16S rDNA sequence (Fig. 2 2A) and the concatenated 16S rDNA, galE, pstC + pstS, and glmS sequences (Fig.2 2B). The SJM1 isolate belonged to the same clade as P. stewartii subsp. indologenes and was 99% homologous to P. stewartii subsp. indologenes strain ZJ-FGZX1 (Fig. 2 2B; Ren et al. 2020). Pathogenicity tests were performed through prick wound inoculation. Sterile needles were used to create fresh wounds on healthy young leaves of one-year-old B. spectabilis plants. Wounds were inoculated with 20 μl bacterial suspension (1 × 108 CFU/ml) or SDW. Four leaves per plant and three plants per treatment were evaluated. The plants were incubated at 28 °C temperature and 80-90% relative humidity. After 4-7 days of inoculation, all plants inoculated with the bacterial suspension had spot symptoms with a halo, similar to those observed in the field. However, leaves inoculated with SDW alone did not show any symptoms. Furthermore, the colony morphology and 16S rDNA sequences of the strains isolated from the inoculated leaves were identical to those of the original isolates. These results verified Koch's postulates. Based on biochemical identification and sequencing analysis, the pathogen causing B. spectabilis leaf spot was identified as P. stewartii subsp. indologenes. Previous reports have shown that P. stewartii subsp. indologenes can cause diseases in Dracaena sanderiana, Cenchrus americanus, and Allium cepa (Zhang et al. 2020, Ashajyothi et al. 2021, Stumpf et al. 2018). To our knowledge, this is the first report of P. stewartii subsp. indologenes causing B. spectabilis leaf spot disease in China.

Phylogenetic Position of Eurihaline Rotifer Brachionus sp. Originated From Tumpaan Waters, South Minahasa, North Sulawesi

This study aims to determine the phylogenetic position of rotifer Brachionus sp. sample originated from Tumpaan coastal water, North Sulawesi in the phylogenetic tree of Brachionus spp. from 27 samples of rotifer selected from 100 samples available in NCBI Genbank. The phylogeny tree was constructed on the basis of COI (Cytochrome Oxidase I) gene sequences using MEGA7 software and the neighbor-joining method. Results show that the phylogeny tree was divided into 2 clades, the group (closely related group) and out-group (distantly related group). It is confirmed that the position of the rotifer originating from Tumpaan Coastal Water is within in group, which has closely related to the B. plicatilis complex BUS06 indicated by a bootstrap value of 80%. The group belongs to SS-type rotifers with an average body size of 149±1.3 μm. This rotifer group has been known as live prey for fish larvae with a mouth-opening size of 150 μm.

Petunia hybrida is a New Host of Pectobacterium brasiliense Associated with Soft Rot Disease in China.

Petunia hybrida is commonly cultivated for ornamental use in urban parks greening and street embellishment in China. In March 2022, 60% of P. hybrida plants cv. Wave Purple (n≈1800) from an ornamental plant nursery under natural conditions in Tianhe district (N 113°21'21", E 23°9'3.5"), Guangzhou, Guangdong Province, China, were affected with soft rot disease. The distribution of the disease was generally uniform. Infected plants initially exhibit small water-soaked lesions at the base of the stem, which then extended to the leaves. Eventually the diseased plant collapsed and died. Nine diseased plants were collected, and affected tissues cut into small fragments (5 × 5 mm), which were disinfested in 75% ethanol (30 s) and 2% sodium hypochlorite (60 s), followed by three rinses with sterile distilled water. The sterilized sections were macerated in 200 μl sterile water, and streaked on Luria-Bertani (LB) agar medium and incubated at 28°C for 48 h. Single colonies were restreaked three times to obtain purified isolation. Sixteen bacterial strains with similar morphology were isolated, and their colonies were yellowish white, round, and convex with smooth surfaces on LB agar plate. The representative strain BDQ1 was selected for further analyses and the 16S rDNA gene (GenBank Accession ON982467) were amplified using primer pair 27F/1492R, revealed above 99% sequence identity with some Pectobacterium brasiliense isolates (GenBank Accession Nos. CP046380(1421/1422), MN393966(1419/1422), and CP020350(1419/1422)) using BLASTn. A multilocus phylogenetic analysis by neighbor-joining method (1,000 bootstrap values) based on six housekeeping gene sequences of gyrA (GenBank Accession No. ON995454), icdA (ON995455), mdh (ON995456), mtlD (ON995457), proA (ON995458), and rpoS genes (ON995459) (Ma et al. 2007; Waleron et al., 2008). The results of phylogenetic analysis showed BDQ1 strain belong to the P. brasiliense clade. Pathogenicity tests were performed on ten healthy P. hybrida cv. Wave Purple plants by injecting 10 µl of bacterial suspensions of BDQ1 (108 CFU/ml) into the stems; another 10 healthy control plants were injected with 10 µl of sterile water. All plants were grown at 25-30°C and 60% humidity in natural light/dark cycle. After 3 d, all inoculated plants showed soft rot symptoms resembling to those observed in the nursery, while control plants remained healthy. Bacteria were successfully reisolated from the symptomatic tissues and identified to be P. brasiliense by PCR mentioned above. P. brasiliense is considered a very aggressive pathogen, which has been reported in Eurasia and Africa (Oulghazi et al. 2021). To our knowledge, this is the first report of P. brasiliense causing bacterial soft rot on P. hybrida in China. This pathogen may pose threat to P. hybrida production in area with warmand humid climate in China. The current study expands the known host range of P. brasiliense and helped raise attention on controlling pathogen spread.

Митохондриальная филогения и систематика орлов Палеарктики

The eagles sensu lato are a polyphyletic group of large raptors represented in the Palearctic realm by 13 species belonging to two subfamilies: Aquilinae (true Eagles) and Haliaeetinae (Sea Eagles). Phylogenetic position of this group taxa at species level and above based on nuclear and mitochondrial markers is currently clarified (Mindell et al., 2018), while intraspecific variability and genetic differences between subspecies are generally not well understood. This study examined the available mitochondrial DNA sequences from GenBank and BOLD databases, also unpublished sequences obtained by the authors were added. Three markers for which sufficient data is available were selected for analysis: cytochrome oxidase 1 (COI) used for DNA-barcoding, cytochrome b (Cyt b), popular in phylogenetic studies, and the hypervariable control region of mtDNA. Phylogenetic trees were constructed using Bayesian Inference, Maximum Likelihood and Neighbor-joining methods, haplotype networks were also constructed. Genetic distances between taxa and time of its divergence were calculated. The obtained results are being discussed in a comparative context at the inter – and intraspecific levels. The possibility of its application and the use of particular genetic markers in general to solve problems of systematics of these species, as well as recent taxonomic changes are considered. The questions of the formation of taxa in the evolution process and their modern distribution in the Palearctic are discussed.

Current understanding of an Emerging Coronavirus using in silico approach: Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2)

Abstract Novel coronavirus (nCoV) namely “SARS-CoV-2” is being found responsible for current PANDEMIC commenced from Wuhan (China) since December 2019 and has been described with epidemiological linkage to China in about 221 countries and territories until now. In this study we have characterized the genetic lineage of SARS-CoV-2 and report the recombination within the genus and subgenus of coronaviruses. Phylogenetic relationship of thirty nine coronaviruses belonging to its four genera and five subgenera was analyzed by using the Neighbor-joining method using MEGA 6.0. Phylogenetic trees of full length genome, various proteins (spike, envelope, membrane and nucleocapsid) nucleotide sequences were constructed separately. Putative recombination was probed via RDP4. Our analysis describes that the “SARS-CoV-2” although shows great similarity to Bat-SARS-CoVs sequences through whole genome (giving sequence similarity 89%), exhibits conflicting grouping with the Bat-SARS-like coronavirus sequences (MG772933 and MG772934). Furthermore, seven recombination events were observed in SARS-CoV-2 (NC_045512) by RDP4. But not a single recombination event fulfills the high level of certainty. Recombination mostly housed in spike protein genes than rest of the genome indicating breakpoint cluster arises beyond the 95% and 99% breakpoint density intervals. Genetic similarity levels observed among “SARS-CoV-2” and Bat-SARS-CoVs advocated that the latter did not exhibit the specific variant that cause outbreak in humans, proposing a suggestion that “SARS-CoV-2” has originated possibly from bats. These genomic features and their probable association with virus characteristics along with virulence in humans require further consideration.

First report of Athelia rolfsii (= Sclerotium rolfsii) causing southern blight on Pachyrhizus erosus in Mexico.

Pachyrhizus erosus, commonly named jicama, is native to Mexico and is cultivated for its tuberous roots which are edible. In November 2021, field sampling was carried out in municipality of Huaquechula (18.748640N, 98.550817W, 1,580 m above sea level), state of Puebla, México. The disease had an incidence between 20 and 30% in approximately 10 ha. Infected plants showed wilting, yellowing foliage, rotting with white mycelium, abundant sclerotia were observed in the roots and tuber. Tuber splits transversely over time. Twenty plants with symptoms of disease were carried out to isolate the fungus. The sclerotia found in the tubers were disinfected with 3% NaOCl, rinsed twice with sterile distilled water, and excess moisture was removed and, transferred on Potato Dextrose Agar (PDA) culture medium and incubated at 28°C. Mycelial fragments from symptomatic tubers, were plated directly to PDA. Twenty representative isolates were obtained by hyphal-tip method, one for each diseased plant sampled (10 isolates from sclerotia and the other 10 from fragments of mycelium). After 10 days, colonies showed fast-growing, dense, cottony-white aerial mycelium, forming globoid to irregular sclerotia, measuring 1.0-1.7 mm in diameter (mean = 1.42 mm; n=100). The number of sclerotia produced per Petri dish ranged from 54 to 542 (mean = 274, n = 50). These sclerotia were initially white and gradually turned brown. Microscopic examination showed septate hyphae with some cells having clamp connections. Based on morphological characteristics, the fungal isolates were identified as Athelia rolfsii (Curzi) CC Tu & Kimbr (Syn: Sclerotium rolfsii Sacc) (Mordue 1974). For molecular identification, a representative isolate (Sr.1), the ITS region was amplified (650 bp) using primers ITS1/ITS4 (White et al. 1990). The obtained sequence (GenBank: ON206899) was subjected to BLAST analysis, where it had 100% identity with A. rolfsii isolates (GenBank: MG836252 and MH517363). Phylogenetic analysis with the neighbor-joining method in MEGAX, grouped the Sr.1 isolate into a common clade with different A. rolfsii isolates. Pathogenicity was confirmed by inoculating 20 tubers detached from healthy P. erosus variety "Criolla de Morelos", into which a portion of mycelium from the Sr.1 isolate was inserted with a sterile wooden stick at one point per tuber. In five tubers, only a sterile wooden stick was inserted as negative controls. The tubers were placed under laboratory conditions with relative humidity close to 100% and a temperature of 28°C. Symptoms like those observed in the field were observed after five days. Control tubers showed no symptoms. Additional pathogenicity tests were performed on 50 plants of 100-day-old P. erosus of the variety "Criolla de Morelos", grown in pots with sterile soil. Ten sclerotia of 10 days old were deposited at the base of the stem, 10 mm below the soil surface; as control treatment only, sterile distilled water was deposited on 20 plants. The plants were placed in a greenhouse (Center for Technological Innovation in Protected Agriculture of the Popular Autonomous University of the State of Puebla), at 28 ± 1°C and 90% of temperature and relative humidity, respectively. After 15 days, all inoculated plants showed symptoms similar to those observed in the field. Control plants showed no symptoms. A. rolfsii was re-isolated from inoculated tubers and stem, fulfilling Koch's postulates. Previously, A. rolfsii was reported in Mexico, causing southern blight on sesame (Hernández-Morales et al. 2018). To our knowledge, this is the first report of Athelia rolfsii causing southern blight on P. erosus in Mexico (Farr and Rossman 2022). This research is important to design management strategies and prevent its spread to other P. erosus-producing areas.

First report of a 16SrXII‐A phytoplasma strain associated with Camelthorn witches’ broom disease in Iran

Camelthorn (Alhagi maurorum, Fabaceae) is an important weed in Iran but in recent years it has also been planted as a crop to provide fodder. In 2021, camelthorn witches’ broom disease was observed in the Seneiee district in Abarkooh county (Yazd province, Iran). The main disease symptoms were little leaf, internode shortening, yellowing and witches’ broom (Fig. 1). The observed disease incidence was up to 10%. Total DNA was extracted from 0.2 g of fresh leaves from both 14 diseased and five asymptomatic plants. DNA samples were tested for phytoplasmas by direct PCR primed by P1/P7 (Lee et al., 1998) and nested PCR using primers P1/P7 and R16F2n/R16R2 (Gundersen & Lee, 1996). PCR amplicons of c. 1.8 and 1.25 Kb respectively were obtained from all 14 diseased Alhagi maurorum plants but not from the symptomless plants. Four R16F2n/R16R2 primed amplicons were sequenced directly. The sequences were identical and a consensus sequence of 1,245 bp was deposited in GenBank (Accession No. OP020342). Sequence comparison by BLAST analysis showed the highest sequence identity with phytoplasma members of the stolbur group (16SrXII). Phylogenetic analysis using the neighbour-joining method (MEGA 7.0) confirmed that the camelthorn phytoplasma clustered within the group 16SrXII phytoplasma clade, closer to strains affiliated to the 16SrXII-A subgroup than to other strains in the same group (Fig. 2). Computer-simulated analysis with 17 restriction endonucleases using iPhyClassifier (https://plantpathology.ba.ars.usda.gov/) showed that the RFLP pattern derived from the camelthorn phytoplasma 16S rRNA gene was identical to “Ca. Phytoplasma solani”, a 16SrXII-A strain (AF248959). This is the first report of a 16SrXII-A phytoplasma associated with camelthorn witches’ broom disease in Iran and to our knowledge, in the world. Previously a 16SrII subgroup C phytoplasma strain was reported to be associated with camelthorn witches’ broom disease in Iran (Esmaeilzadeh-Hosseini & Salehi, 2021). Since 16SrXII-A subgroup phytoplasmas have been detected previously in grapevine in Fars and Lorestan (Salehi et al., 2014) and Yazd (Salehi et al., 2016) provinces, it is suggested that camelthorn can serve as a reservoir for 16SrXII-A phytoplasmas.

First report of a ‘Candidatus Phytoplasma aurantifolia’ related strain (16SrII‐C) associated with Convolvulus virgatus witches'‐broom disease in Iran

Convolvulus virgatus (Convolvulaceae) is a perennial shrub which grows mainly in desert and shrubland biomes in Iran, the Arabian Peninsula and Pakistan (Wood et al., 2015). In a 2020 survey for phytoplasma diseases, witches'-broom disease of C. virgatus (WBDCV) was observed in the Roodan area (Hormozgan province, Iran). The main symptoms of WBDCV were flower and stem proliferation, shortened internodes, little leaf, severe witches'-broom, yellowing, reddening, plant sterility and decline (Figs. 1, 2). Total DNA was extracted from leaves and leaf petioles of five symptom-bearing and five symptomless C. virgatus plants using a CTAB-based method. DNA samples were tested for the presence of phytoplasmas by direct-PCR using primer pair P1/P7 (Lee et al., 1998) and nested PCR using primer pairs P1/P7 and R16F2n/R16R2 (Gundersen & Lee, 1996). The expected amplicons of c. 1,800 bp and 1,250 bp were obtained in direct and nested-PCR respectively, from all diseased plants but not symptomless ones. Two R16F2n/R16R2 amplicons were randomly selected and cloned into Escherichia coli DH5α cells using the PCR TA Cloning Kit (SinaClon, Cat. No.:CL5841, Iran) and three plasmid DNAs from recombinant colonies were sequenced. The obtained sequences were identical, and a consensus sequence was deposited in GenBank (Accession No. ON361565). BLASTn analysis of the obtained sequence showed the highest sequence identity (98.7-99.9 %) with phytoplasmas related to ‘Ca. P. aurantifolia’ (16SrII group). Phylogenetic analysis with MEGA11 software using the neighbour-joining method Fig. 3 revealed that the WBDCV phytoplasma clustered within the ‘Ca. P. aurantifolia’ (16SrII) group clade, closest to the strains of subgroup 16SrII-C. In Iran, phytoplasma strains belonging to 16SrII-C subgroup have been reported previously in Bushehr, Fars (adjacent to Hormozgan province) and Yazd provinces in several plant species including Daucus carota, Medicago sativa, Prunus dulcis, Vicia faba (Siampour et al., 2019), Alhagi maurorum and Artemisia sieberi (Esmaeilzadeh-Hosseini & Salehi, 2021); however, this is the first report of a ‘Ca. P. aurantifolia’-related strain in C. virgatus associated with WBDCV in Iran and most probably, in the world. The authors would like to thank the head of the Fars Agricultural and Natural Resources Research and Education Centre, Shiraz, Iran, for support and encouragement.

Carica papaya Represents a New Host of 16SrI-B Subgroup Phytoplasma Associated with Yellow Symptoms in China.

Carica papaya Linn, belonging to the Caricaceae family, is an economic and medicinal plant, which is widely cultivated in tropical and subtropical countries (Soib et al., 2020). Beginning in 2021, abnormal symptoms of Carica papaya exhibiting leaf yellow, crinkle and leaflet were found in Wanning city of Hainan Province, China. The diseased symptoms of the plant, with about 20 % incidence in the sampling regions, were suspected to be induced by phytoplasma, a phloem-limited and could not be cultured in vitro prokaryotic pathogen. Total DNAs were extracted from 0.10 g fresh leaves of symptomatic or asymptomatic Carica papaya using CTAB DNA extraction method (Doyle and Doyle, 1990). PCR reactions were performed using primers R16mF2/R16mR1 (Gundersen and Lee, 1996), secAfor1/secArev3 (Hodgetts et al., 2008) and AYgroelF/AYgroelR (Mitrović et al., 2011) specific for phytoplasma 16S rRNA, secA and groEL gene fragments. PCR products of the 16S rRNA, secA and groEL gene target fragments of phytoplasma were obtained from the DNA of eight diseased Carica papaya samples whereas not from the DNA of the asymptomatic plant samples. The PCR amplicons of the three genes were cloned and sequenced by Biotechnology (Shanghai) Co., Ltd. (Shanghai, China) and the sequences data were deposited in GenBank. The 16S rRNA, secAgroEL gene of phytoplasma was in length of 1326 (GenBank accession: OL625608), 716 (OL630087) and 1300 (OL630088) bp separately, putatively encoding 238 (secA) and 432 (groEL) amino acids sequence. The phytoplasma strain was named as Carica papaya yellow phytoplasma (CpY), CpY-hnwn strain. A blast search based on 16Sr RNA gene of CpY-hnwn showed 100 % sequence identity with that of 16SrI aster yellows group members (16SrI-B subgroup), such as Onion yellows phytoplasma strain OY-M (AP006628), Chinaberry witches'-broom phytoplasma (CWB) strain CWB-hnsy1 (KP662119) and CWB-hnsy2 (KP662120), Rapeseed phyllody phytoplasma isolate RP166 (CP055264). RFLP analysis based on the 16S rRNA gene fragment of CpY-hnwn was performed by the interactive online phytoplasma classification tool iPhyClassifier (Zhao et al., 2009) indicated that the phytoplasma strain is a member of 16SrI-B subgroup. Blast search based on secA gene of CpY-hnwn showed 100 % sequence identity with that of CWB strains CWB-gdgz (KP662182), CWB-jxnc (KP662180) and CWB-fjya (KP662178) belonging to 16SrI-B subgroup. Blast search based on groEL gene of CpY-hnwn showed 99.77 % sequence identity with that of mulberry dwarf phytoplasma (AB124809) and 99.69 % sequence identity with that of Onion yellows phytoplasma strain OY-M (AP006628) and Rapeseed phyllody phytoplasma isolate RP166 (CP055264). Phylogenetic analysis based on the 16S rRNA gene fragments performed by MEGA 7.0 employing neighbor-joining (NJ) method with 1000 bootstrap value (Kumar et al., 2016; Felsenstein, 1985) indicated that the CpY-hnwn phytoplasma strain was clustered into one clade with the phytoplasma strains of OY-M (AP006628), RP166 (CP055264), CWB-hnsy1 (KP662119), CWB-hnsy2 (KP662120) and areca palm yellow leaf (KF728948), with 100 % bootstrap value. To our knowledge, this is the first report that a 16SrI-B subgroup phytoplasma infects Carica papaya in Hainan Province, a tropical island of China. Carica papaya was previously reported to be infected by 16SrXII-O subgroup phytoplasmas in Nigeria (Kazeem et al., 2021), 16SrII-U subgroup in Hainan Province of China (Yang et al., 2016). The findings in this study indicated that one plant couldthe phytoplasmas belonging to different 16Sr groups, which would be beneficial to the specific detection of the pathogens and the epidemic monitoring of the related diseases.

Prevalence of Tri­cho­monas gallinae with molecular characterisation and phylogenetic analysis in domestic pigeons using ITS1-5.8s rRNA-ITS2 gene in Alborz province, Iran

Avian trichomonosis is an important parasitic disease throughout the world caused by the protozoan Trichomonas gallinae, commonly seen in pigeons and wild birds. Lesions in the upper gastrointestinal tract (beak and crop) are complications of this disease. Currently, the diagnosis of this organism is made using laboratory methods including direct smear, culture medium and molecular methods. The aim of the present study was to survey the prevalence of T. gallinae in pigeons of Alborz province, Iran through molecular and phylogenetic identification using ITS1-5.8s rRNA-ITS2 gene. A total of 87 samples were collected from domestic pigeons from May to September 2019. The samples were taken directly from the mouth and larynx using an oral swab. Out of 87 collected samples, 28 (32.18 %) were positive using direct smear, culture and polymerase chain reaction (PCR) methods. Based on the results, the size of the amplification product of this gene was 372 base pairs. The results of this study were analysed using a phylogenetic tree and Neighbour-Joining (NJ) method. The present study showed high prevalence of T. gallinae in pigeons. Two types of T. gallinae genotypes, A and B, were found in pigeons. Also, the phylogenetic analysis of ITS1-5.8s rRNA-ITS2 sequences from positive samples, showed high coverage with sequences present in the GenBank.

Genotypic diversity and abiotic stress response profiling of short-grain aromatic landraces of rice (Oryza sativa L. Indica)

This study evaluated the physiological responses and genetic diversity of selected short-grain aromatic rice landraces of Northern Eastern Ghats of India to multiple abiotic stresses. Results revealed that drought, salt and flooding stress remarkably declined plant biomass, relative growth index (RGI), relative water content (RWC), leaf photosynthesis, PSII activity, SPAD relative index, and elevated effect was found in susceptible IR64 (susceptible check) variety. We employed molecular marker analysis to characterize 21 rice genotypes using 32 SSR primers related to drought, flooding and salinity tolerance QTLs. Our analysis suggests significantly high polymorphism (93.12%) in 96 loci. The mean value of polymorphism information content (PIC), marker index (MI) and resolving power (RP) and heterozygosity index (HI) were 0.326, 1.037, 1.558 and 0.420 respectively. The Jaccard’s similarity coefficient ranged from 0.158 to 0.846 with a mean genetic dissimilarity of 0.647. According to neighbor-joining method of clustering, all genotypes were grouped into two major and five sub-clusters which is concurrent with a very broad genetic base (K = 5) obtained with STRUCTURE analysis. Taken together, six aromatic genotypes Basubhoga, Dudhamani, Kalajeera, Laktimachi, Kuyerkuling and Tulasi are genetically very close to drought-tolerant (N22) and flooding tolerant (FR13A) cultivar whereas, salt-tolerant (Pokkali) very close to Sapuri and Muktabali. The degree of genetic variants obtained from this profiling could be useful for landrace conservation, selection and global implications for multiple abiotic stress tolerance.