Sequence Characterized Amplified Region Research Articles

Molecular Identification and Scar Detection of Entomopathogenic Fungi of the Genus Beauveria

In recent years, there has been increasing interest in identifying and studying beneficial microorganisms that can be used in organic farming as fertilizers and plant protection agents. Entomopathogenic fungi with an endophytic nature of the genus Beauveria are quite promising and their identification and detection in the plant and soil samples are of great importance. Molecular identification of fungi of the genus Beauveria was conducted by sequencing the internal transcribed spacer 1 and 5.8S ribosomal RNA gene (ITS) regions with universal primers. As a result of the sequencing, it was found that the analysed strains belong to four genera of Beauveria with the following percentage distribution: B. bassiana (68.18%), B. pseudobassiana (13.64%), B. varroae (9.09%) and B. tenella (9.09%). The largest percentages of the strains were identified as B. bassiana. Sequence-characterized amplified regions (SCAR) have been applied for the detection of Beauveria strains in the field samples. The efficiency and ability of three pairs of SCAR markers to differentiate the studied strains were tested. The applied SCAR markers generated fragments with expected length in more than 90% of the investigated strains and could be used as markers for the detection of the fungi from the genus Beauveria in field samples. In the selected experimental conditions only B. varroe 501 and B. tenella 593 lack fragments with used SCAR markers.

Morphological and Molecular Identification of Root-knot Nematode, Meloidogyne incognita Infecting Pomegranate (Punica granatum L.) in Jodhpur, Rajasthan, India

Root-knot nematodes (Meloidogyne spp.) pose a major threat to pomegranate (Punica granatum L.) cultivation in India, leading to significant yield losses. In this study, galled roots of pomegranate were collected from orchards in Jodhpur, Rajasthan and nematode infestation was confirmed through root staining. The perineal pattern of females displayed the typical characteristics of the Meloidogyne incognita. To further confirm the species, DNA was extracted and a polymerase chain reaction (PCR) assay using species-specific SCAR (sequence-characterized amplified region) primers was conducted. The M. incognita- species specific primers, MincF1/MincR1, yielded the expected 150 bp product, verifying the presence of M. incognita. This study marks the first molecular confirmation of M. incognita infecting pomegranate orchards in this region, demonstrating the efficacy of SCAR markers in complementing traditional morphological identification as well.

Development and Molecular Characterization of a Sequence Characterized Amplified Region (SCAR) Marker for the Identification of Hybrid Oil Palm (Elaeis guineensis Jacq.)

Development and Molecular Characterization of a Sequence Characterized Amplified Region (SCAR) Marker for the Identification of Hybrid Oil Palm (<i>Elaeis guineensis</i> Jacq.)

Rapid Diagnostic PCR Assay Method for Species Identification of Mantidis Ootheca (Sangpiaoxiao) Based on Cytochrom C Oxidase I (COI) Barcode Analysis.

Mantidis Ootheca (sangpiaoxiao), the egg case of the mantis, is a type of insect-derived traditional medicine widely used in East Asia. However, species identification based on egg morphology is challenging, leading to the distribution of counterfeit and adulterated products. The use of inauthentic ingredients can pose serious health risks to consumers. This study aimed to develop PCR markers that can rapidly and accurately differentiate between authentic and counterfeit Mantidis Ootheca. The mitochondrial cytochrome c oxidase I (COI) region was sequenced in thirteen samples from four mantis species: Tenodera angustipennis, Statilia maculata, Hierodula patellifera, and T. sinensis. Four sets of SCAR primers were designed based on species-specific nucleotide polymorphisms, and a multiplex SCAR assay was developed by combining all sets of the primers. The sequence-characterized amplified region (SCAR) primers successfully produced amplicons for each target species, even with low-DNA templates or templates containing DNA from multiple samples. No amplification was observed for nontarget species. This study presents a novel approach for identifying authentic Mantidis Ootheca species using DNA-based diagnostic marker assays, which enable rapid and precise species identification. The SCAR assays developed in this study will aid in maintaining quality control and promoting the standardization of commercial Mantidis Ootheca products.

Identification of a major QTL region and candidate genes responsible for harvest date combining QTL mapping and QTL-seq approaches in walnut (Juglans regia L.)

Walnut (Juglans regia L.) takes place within the Juglandaceae family and is one of the most popular nut species. In walnut breeding, several limiting factors need to be revealed regarding the genetic mechanism underlying important agronomical traits. In the current report, the monitoring of the ‘Chandler x Kaplan-86’ F1 walnut population was carried out four successive years (2015, 2016, 2017, 2018) and the distribution of the plants for harvest date from four years was ranged from August 27th to October 22nd. Then, we dissected the genomic regions associated with harvest date using classical Quantitative Trait Loci (QTLs) mapping and a rapid and cost-effective QTL-seq approach in ‘Chandler x Kaplan-86’ F1 walnut population for the first time. In classical QTL mapping, a major QTL was recognized in LG4 corresponding to Chr1 in Chandler female map with 27.6 (%) phenotyping variance. In QTL-seq, we bulked the DNAs of the genotypes with extreme harvest date separately, the whole genome resequencing was performed for a parent Chandler and both bulks (early and late) to calculate Delta SNP indexes for each Single Nucleotide Polymorphisms (SNPs) and Insertion/Deletion (InDels). Approximately 48.04 million paired reads for Chandler, 149.72, and 150.14 million paired reads were obtained from early and late harvest bulks, respectively. A total of 229,307 SNPs and 45,223 InDels were mined from the merged sequences data covered whole chromosomes. The responsible genomic fragments were identified in Chr2 and Chr11 in QTL-seq. The highest linked InDel locus detected in QTL-seq in Chr11 around 17 million base pairs distinguished the plants according to harvest date. InDel Sequence Characterized Amplified Region (SCAR) marker is a candidate for marker-assisted selection for future walnut breeding studies.

Tilletia laevis caused common bunt disease of wheat in district Swat, Khyber Pakhtunkhwa-Pakistan

Wheat common bunt caused by Tilletia laevis (Kühn) and T. caries (Bjerk.) Wint., is one of the world's most destructive diseases (He et al., 2022). In June 2022, we observed wheat common bunt symptoms in ten fields of city Mingora in Swat District, Khyber Pakhtunkhwa, Pakistan (35.2227°N, 72.4258°E) with spring cultivar Galaxy 2013. The corresponding disease heads were collected to isolate and characterized the causal agent of wheat common bunt. Teliospores morphology, germination, and disease symptoms revealed that fungal isolates belong to Tilletia laevis, which cause common bunt disease of wheat. After Koch's postulates, the artificially inoculated plants showed similar symptoms to those observed in the fields with same teliospores morphology and germination. Tilletia laevis was also confirmed through molecular characterization using sequence characterized amplification region (SCAR) markers specific for T. laevis and obtained 660 bp bands from leaves, stem and heads from artificially inoculated plants. Therefore, to the best of our knowledge, this is the first report of T. laevis causing common bunt in Mingora, Swat, Khyber Pakhtunkhwa, Pakistan.

Inter-specific hybrids in Areca spp.: Verification using SCoT markers

Disease resistance has been a major goal in crop improvement programmes in arecanut (Areca catechu L.), where the fruit rot diseaseor Mahali, caused by Phytophthora meadii, is a major production constraint in India. The wild Areca spp., such as A. triandra and A.concinna, have been reported to possess resistance to P. meadii. Developing inter-specific hybrids between A. catechu and A. triandraor A. concinna could be one of the strategies to introgress the disease-resistant trait from the wild Areca spp. into cultivated arecanut.In this study, we report the utilization of start codon targeted (SCoT) markers to differentiate A. catechu from A. triandra and A. concinnaand the development of sequence-characterized amplified region (SCAR) markers to enable authentication of true inter-specific hybridsbetween them. The technique would effectively verify inter-specific hybrids at the seedling stage itself.

The development and utilization of two SCAR markers linked to the resistance of banana (Musa spp. AAA) to Fusarium oxysporum f. sp. cubense race 4

Banana (Musa spp.) production worldwide is seriously threatened by Fusarium wilt caused by Fusarium oxysporum f. sp. cubense (Foc). The best way to control this disease is to grow resistant cultivars. However, it requires large-scale field evaluations and labor- and time-consuming to obtain disease-resistant germplasm. Development of early, reliable, and reproducible selection strategies are considered as the efficient approach which could speed up the selection procedure. In this study, two pairs of sequence-related amplified polymorphism (SRAP) primers related to banana Foc resistance/susceptibility were screened from 100 pairs of random primers. Correspondingly, two pairs of sequence characterized amplified region (SCAR) markers (namely SC4-F/SC4-R and SC14-F/SC14-R, respectively) were successfully generated from these two SRAP markers using 30 cultivars either resistant or susceptible to Foc. Both SCAR markers were located in mitochondrion genome and showed discriminatory power of 96.67% and 100%, respectively. Mitochondrial proteins possibly play a very important role in banana resistance to Foc. In additional, these two SCAR markers were employed simultaneously to screen potential resistant germplasm from 53 accessions with unknown resistance to Foc, and the results revealed a consistency of 83.0% with each other, further indicating their high reliability and reproducibility. These results suggest that both SCAR markers could be used in molecular marker-assisted selection for banana germplasm resistant to Fusarium.

Identification and Biological Characterization of a New Cyst Nematode, Heterodera glycines sbsp.n. tabacum, Parasitizing Tobacco in China.

In an investigation of diseases from plant-parasitizing nematodes in Henan Province, a cyst nematode was found on tobacco roots and in rhizosphere soil. We identified this strain as a new cyst nematode subspecies, Heterodera glycines sbsp.n. tabacum. The cysts and second-stage juveniles (J2s) parasitizing Henan tobacco were larger than those of H. glycines. A single 345-bp fragment was amplified from H. glycines sbsp.n. tabacum, whereas the 345- and 181-bp fragments were amplified from the soybean cyst nematode. Thus, H. glycines sbsp.n. tabacum was distinct from H. glycines. There were base transversions at 504 sites and base transitions at 560, 858, 920, and 921 sites in the rDNA-ITS sequences of H. glycines sbsp.n. tabacum compared with H. glycines, and there were base transitions at 41, 275, 278, and 380 sites in the mtDNA-COI sequences. In the phylogenetic tree based on the rDNA-ITS and mtDNA-COI regions, H. glycines sbsp.n. tabacum was clustered on a single branch. Based on the randomly amplified polymorphic DNA (RAPD) technique, sequence characterized amplified region (SCAR)-PCR primers were designed. A single 1,113-bp fragment was amplified by specific primers (HtF1/HtR1) from H. glycines sbsp.n. tabacum, while no fragments were obtained from H. glycines. The H. glycines sbsp.n. tabacum can infect soybean plants but cannot complete its life cycle on soybean. Eleven tested tobacco cultivars were infected, with an average reproduction factor (Rf) of 9.74 and a maximum of 64.2 in 'K326'. The cumulative egg hatching rate of H. glycines sbsp.n. tabacum in the presence of tobacco root exudates was 42.6% at 32 days posthatching, which was significantly greater than that in the presence of soybean root exudates (30.3%) or sterile water (33.1%). In summary, the cyst nematode population parasitizing Henan tobacco was identified as a new subspecies, H. glycines sbsp.n. tabacum.

Unveiling mechanisms for induced systemic resistance, resistance breeding and molecular marker‐assisted breeding against Phomopsis blight of Solanum melongena

AbstractPhomopsis vexans is one of the most destructive fungal pathogens associated with eggplant and currently poses a significant threat to eggplant production worldwide. The detrimental impact of P. vexans on eggplant yield has been extensively explored by various mycologists who have conducted thorough studies on the diversity, pathology and biological aspects of the pathogen. However, achieving enduring resistance or effective management has proven to be a challenge thus far. PCR‐based detection and molecular association of Phomopsis resistance use molecular markers to examine the potential for heterosis in various crosses, aiming for premium hybrids with genetic resistance and high‐yielding capabilities. The latest genome sequencing methods and availability of a wider range of genetic diversity has enabled the breeding of resistant varieties of eggplant. This review provides a detailed description on P. vexans including its epidemiology, dispersal methods, symptomology, colony characteristics, taxonomy and evolution of its strains. Different resistance breeding techniques including heterosis breeding, host plant resistance, identification of resistant sources, inheritance pattern for Phomopsis resistance, importance of grafting to impart resistance, significance of induced resistance, PCR‐based detection and molecular association of Phomopsis resistance are explained. Future approaches include molecular marker techniques such as genome‐wide association studies, sequence‐characterized amplified regions (SCAR), role of biotic inducers, pathogenesis‐related proteins and plant growth‐promoting rhizobacteria against Phomopsis blight of eggplant.

Development of highly discriminatory SCoT- and CBDP-based SCAR fingerprint for authentication of Indian senna (Senna alexandrina Mill.) formerly Cassia angustifolia Vahl.).

Indian senna (Senna alexandrina Mill.) (formerly Cassia angustifolia Vahl.) is an important medicinal plant of the family Fabaceae. The leaves and pods of Indian senna yield sennosides and rhein-based laxative. Adulteration of Indian senna is a serious issue as with most of the medicinal plants used in the Indian systems of traditional medicine. The bulk of dried leaves and pods of morphologically related species, such as Cassia fistula, Senna occidentalis, Senna sophera, and Senna tora, is usually mixed with those of the Indian senna, and the admixture is used in laxative-based formulations. The present investigation is a modest attempt at developing species-specific start codon targeted (SCoT) polymorphism- and CAAT-box-derived polymorphism (CBDP)-based sequence-characterized amplified region (SCAR) markers for the identification and authentication of Indian senna and four adulterant species (C. fistula, S. occidentalis, S. sophera, and S. tora species). In this study, genomic DNA extracted from 44 accessions of Indian senna and four adulterant species was subjected to SCoT and CBDP PCR. The polymorphic amplicons were identified, eluted, ligated, and transformed into Escherichia coli DH5 α strain. PCR, restriction analysis, and DNA sequencing confirmed the transformed recombinant plasmid clones. Post-sequencing, the sequence of the primary SCoT and CBDP primers was analyzed and extended into the unique signature sequence of the concerned accessions. This resulted in development of one SCoT-44- and two CBDP-25-based SCARs. SCoT-44 SCAR produced a signature amplicon of 287 bp for accession DCA120, and CBDP-25 SCAR yielded signature amplicons of 575 and 345 bp for accessions DCA13 and DCA119, respectively. The developed SCAR markers were validated across 48 samples (44 accessions of Indian senna and 4 adulterant species) and produced distinct amplicons in Indian senna only, while no such amplicon was observed in the other four adulterant species. The information generated using these markers have been faithfully converted to single-locus, unequivocal, highly reproducible, and informative sequence-based SCAR markers. These markers will enable discrimination of individual plants on the basis of unique sequence-specific amplicons, which could be used as diagnostic markers to settle issues pertaining to the true identity of Indian senna.

Advances in Physalis molecular research: applications in authentication, genetic diversity, phylogenetics, functional genes, and omics.

The plants of the genus Physalis L. have been extensively utilized in traditional and indigenous Chinese medicinal practices for treating a variety of ailments, including dermatitis, malaria, asthma, hepatitis, and liver disorders. The present review aims to achieve a comprehensive and up-to-date investigation of the genus Physalis, a new model crop, to understand plant diversity and fruit development. Several chloroplast DNA-, nuclear ribosomal DNA-, and genomic DNA-based markers, such as psbA-trnH, internal-transcribed spacer (ITS), simple sequence repeat (SSR), random amplified microsatellites (RAMS), sequence-characterized amplified region (SCAR), and single nucleotide polymorphism (SNP), were developed for molecular identification, genetic diversity, and phylogenetic studies of Physalis species. A large number of functional genes involved in inflated calyx syndrome development (AP2-L, MPF2, MPF3, and MAGO), organ growth (AG1, AG2, POS1, and CNR1), and active ingredient metabolism (24ISO, DHCRT, P450-CPL, SR, DUF538, TAS14, and 3β-HSB) were identified contributing to the breeding of novel Physalis varieties. Various omic studies revealed and functionally identified a series of reproductive organ development-related factors, environmental stress-responsive genes, and active component biosynthesis-related enzymes. The chromosome-level genomes of Physalis floridana Rydb., Physalis grisea (Waterf.) M. Martínez, and Physalis pruinosa L. have been recently published providing a valuable resource for genome editing in Physalis crops. Our review summarizes the recent progress in genetic diversity, molecular identification, phylogenetics, functional genes, and the application of omics in the genus Physalis and accelerates efficient utilization of this traditional herb.

Discrimination of lethal Russula subnigricans from wild edible and morphologically similar mushrooms in the genus Russula using SCAR markers

Russula is a diverse genus of mushrooms that is consumed worldwide. Russula subnigricans is a lethal species that causes myotoxicity upon ingestion. It is morphologically similar to wild foraged species, including R. nigricans and R. densifolia. Misidentification of these mushrooms can lead to severe food poisoning. Several PCR-based approaches have been used to overcome this problem. In this study, 22 samples of wild edible Russula species were collected, and five clinical samples of R. subnigricans leftovers were obtained. Based on the sequence data of the internal transcribed spacer, the phylogenetic trees revealed that these 27 samples could be classified into well-established clades of six subgenera. Fifty arbitrary primers were used in high annealing temperature rapid amplified polymorphic DNA (HAT-RAPD) technique to screen polymorphic bands among R. subnigricans, R. nigricans, R. densifolia, and other edible species. Distinctive RAPD fragments were obtained from 11 primers and used in sequence characterized amplified region (SCAR) technique. Three pairs of species-specific SCAR primers were selected to distinguish lethal R. subnigricans, R. nigricans, and R. densifolia with SCAR markers of 213, 420, and 609 bp, respectively. Ten-fold sensitivity test revealed that the SCAR marker of R. subnigricans could be detected at DNA template concentrations as low as 30 pg/μL. This marker could detect the presence of lethal R. subnigricans even at 1% in a cooked sample of mixed mushroom remnants. The developed SCAR markers ensured rapid and accurate diagnosis of suspected myotoxic mushrooms, thereby reducing the risk of severe foodborne illness and death.

Characterization of Trichoderma strains for novel species-specific markers by multiplex PCR and antagonistic property against Alternaria ricini in castor (Ricinus communis L.)

The study aimed at characterizing six Trichoderma strains isolated from the rhizospheric soil of castor growing region of Telangana, India. Microscopic examination and sequencing of ribosomal DNA (rDNA) using internal transcribed spacer (ITS) and D1-D2 region-specific primers revealed them as members of Trichoderma sp. Whereas the phylogenetic analysis revealed four of those strains as Trichoderma asperellum, two strains were confirmed as Trichoderma harzianum with a high sequence similarity of >99%. Further, multiplex PCR results with Trichoderma-specific primers; TEF1 (translation elongation factor 1) and rpb2 (the second largest subunit of RNA polymerase II) gene-specific primers confirmed the findings resulting from the phylogenetic analysis. To develop a set of sequence characterized amplified region (SCAR) markers, these strains were screened with 185 random amplified polymorphic DNA (RAPD) primers. A pairwise comparison of correlation of coefficient values determined from the RAPD-based genetic similarity analysis revealed that TV3 and TS4 strains were closely related to each other (0.98) confirming them as T. harzianum while N13, 7316, TV5, and TV2B16 strains formed a different cluster with T. asperellum strains. A set of novel SCAR markers were developed to distinguish these two species, T. asperellum and T. harzianum including the reported Trichoderma strains as Ex-type strains. T. harzianum strains had extremely low sporulation ability to be tested for biocontrol properties. Of the four T. asperellum strains, 7316 followed by N13 demonstrated high antagonistic ability against the fungal pathogen, Alternaria ricini indicating these strains as potential biocontrol agents to limit Alternaria infection in castor.

Development of a sequence-characterized amplified region (SCAR) marker for female off-season flowering detection in date palm (Phoenix dactylifera L.)

Development of a sequence-characterized amplified region (SCAR) marker for female off-season flowering detection in date palm (<i>Phoenix dactylifera</i> L.)

DNA barcodes, ISSR, RAPD and SCAR markers as potential quality control tools for molecular authentication of black and white mulberry

Morus species, especially black mulberry (Morus nigra L.), are well known for their nutritive and economic values. Black mulberry is often a subject for deliberate and undeliberate substitution with the morphologically similar white mulberry (Morus alba L.). In the current study, the genetic diversity of Morus nigra L. and Morus alba L. was assessed using different genetic profiling and fingerprinting techniques namely namely DNA barcoding, Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR). DNA barcoding was implemented using 2 plastid loci (matK, and rbcL-1) and 2 nuclear spacers (ITS and ITS2) aiming at accurate authentication of the studied species. DNA barcoding, targeting the selected loci succeeded to identify and authenticate the studies Morus species but failed to differentiate them, owing to their close genetic resemblance. Out of 52 screened RAPD primers, 27 were able to produce reproducible and clear profiles with 115 amplified fragments of which 37 were polymorphic accounting for 32.17% polymorphism. The number of amplified bands varied from 2 to 7 ranging in size from 100 to 700 bp. Moreover, 55 ISSR primers were evaluated, 108 fragments were produced, using 20 primers, of which 28 were polymorphic accounting for 25.92% polymorphism with size range of 200–800 bp. Sequence Characterized Amplified Region (SCAR) marker was developed based on reproducible banding pattern obtained by RAPD primer (OPG-03), for the purpose of accelerating the initial screening of Morus batches and enabling the preventative rejection of adulterated samples. The polymorphic band obtained was sequenced and specific primers were designed to develop a SCAR marker of approximately 558 bp exclusively for M. nigra, allowing detection of down to 1 ng, of M. nigra. The SCAR marker showed 100% success in differentiating authentic M. nigra and M. alba samples. In addition, the application of the developed SCAR marker was extended to distinguish M. nigra from its four common adulterants namely Ficus carica L., Fragaria vesca L., Ficus racemosa L. and Vitis vinifera L.. The proposed protocol was successfully implemented for the evaluation of 47 commercial M. nigra samples collected from different Egyptian locations.

Development of Local Breeding Lines for Rust Resistance in the Common Bean (&lt;em&gt;Phaseolus vulgaris&lt;/em&gt;) through the Incorporation of Rust-Resistant Genes

Bean rust is one of the major diseases of the common bean (Phaseolus vulgaris) reported in Sri Lanka and at the global level. This study aimed to develop rust-resistant snap bean breeding lines via gene pyramiding assisted by molecular markers. Resistant sources; PI 181996, BelMiNeb-RMR-8, and BelDakMi-RMR-19, enriched with the rust-resistant genes Ur-3 and Ur-11, were selected as donor parents to obtain a wide range of resistance to the rust pathogen. Resistant genotypes were crossed with popular local varieties Kappetipola nil and Galpalama Kalu (Capri) to introgress Ur-3 and Ur-11 resistant genes. Successive F1, F2, and BC (backcross) generations were obtained with the self-pollination and backcrossing processes. Standard phenotypic disease screening methods were applied to identify resistant lines. Phenotypically resistant plants obtained from these crosses were tested with sequence-characterized amplified region (SCAR) markers linked to two rust-resistant genes: SK 14 (linked to Ur-3) and SI 19 (linked to Ur-11). Molecular marker SI-19 showed higher reproducibility (50% to 80%) with the availability of relevant banding patterns for phenotypically resistant F1, F2, and BC1 progenies. However, SK 14 showed lower reproducibility (30–60%) for the same progenies. Approximately 450 genotypes introgressed with rust-resistant genes (Ur-3 and Ur-11)were produced. Among them, four advanced resistant lines obtained from the different cross combinations (Kappetipola nil x BelDakMi-RMR-19, Galpalama Kalu x BelMiNeb RMR-8, Kappetipola nil x PI 181996, and Kappetipola nil x BelMiNeb RMR-8) with preferred agronomic characters were selected for further variety development. All new genotypes will be important for future bean-resistant breeding programs in Sri Lanka.

Diagnosis and Characterization of Ditylenchus destructor Isolated from Mazus japonicus in China.

The potato rot nematode (Ditylenchus destructor) is one of the most destructive pests in the production of tuber crops, resulting in severely decreased yields and inferior product quality. In 2021, a great number of nematodes were detected in the roots of Mazus japonicus, a weed that is harmful to crop growth, in Qingdao, Shandong Province, China. The present study was undertaken to characterize and identify the nematodes isolated from M. japonicus through morphological identification and molecular approaches. Their morphological characteristics were highly consistent with the descriptions of D. destructor Thorne, 1945. The nematodes collected from M. japonicus were identified as D. destructor haplotype B using D1/D2 and sequence characterized amplified region (SCAR) primers. PCR-ITS-RFLP analysis was conducted to monitor intraspecific variations. In addition, the phylogenetic analysis of the internal transcribed spacer (ITS) demonstrated that this D. destructor population was clustered in haplotype B, supported by a 100% bootstrap value. Another assay, in which M. japonicus was inoculated with a mixture of the life stages of D. destructor, was performed. This assay showed that M. japonicus exhibited a high susceptibility to D. destructor in pots. This is the first record of D. destructor parasitizing M. japonicus in China, and it is of great importance because M. japonicus could be a potential reservoir for D. destructor in the field.

First Report of Root-Knot Nematode (Meloidogyne incognita) on Dioscorea opposita in Henan Province, China.

Dioscorea opposita is an annual twining plant in China that is used for consumption and medicinal purposes. The planting area of D. opposita is near 500,000 hectares in China, mainly in Shangdong, Hebei, Henan, Jiangxi and Yunnan provinces. In August 2021, we observed that some D. opposita plants grew poorly with smaller and chlorotic leaves in Changyuan (35°8'12"N; 114°43'52"E), Henan Province, China. Galls with hook-shaped roots and tuber damage were also observed, typical of root-knot nematode. Thirty tubers were randomly collected and 60% were infested with root-knot nematodes. During a disease survey in Changyuan, the incidences of root-knot nematode damage were 31.5%, 21%, and 18% in three fields (0.33, 0.67, and 4 ha, respectively) at harvest. The average tuber length of infected plants was decreased by 65.8%, and the average weight was decreased by 70.1% compared to the healthy plants. Males, females, second-stage juveniles (J2s), and eggs were extracted from individual diseased tubers from the three fields for morphological identification. Females were white, pear-shaped with a projecting neck. Males showed a trapezoidal labial region with prominent stylet knobs, including a high head cap which had a stepped outline and was centrally concave in lateral view. Morphological measurements are described in the supplementary material. All data and descriptions conformed to the morphological characteristics of Meloidogyne incognita. Genomic DNA was extracted from J2s (n=9) using PCR lysis buffer, and used for PCR amplification of the sequence characterized amplified region (SCAR) markers specific for M. incognita. Two pairs of the SCAR primers, Mi-F/Mi-R, and Inc-K14-F/Inc-K14-R, were used to diagnose whether these nematodes from D. opposita were M. incognita (Meng et al. 2004; Randig et al. 2002). The PCR produced expected amplification products of 955 and 399 bp, confirming the nematode to be M. incognita. Primers specific for M. arenaria (Far/Rar) and M. javanica (Fjav/Rjav) were used but failed to amplify fragments (Randig et al. 2002; Zijlstra et al. 2000). The obtained PCR fragments were sequenced and deposited in GenBank (accession no. OQ420602.1, OQ427638.1). They showed 99.9 and 100% identity to the available GenBank M. incognita sequence (accession no. MK410954.1, ON861825.1), respectively. A pathogenicity test was conducted in greenhouse conditions. Bulbils of D. opposita were sown in the pots filled with 2,000 ml of autoclaved soil mixture (loamy soil/sand, 1:1). One month later, 15 seedlings (five to six leaf stage) were inoculated with 1,000 M. incognita J2s individually. Five plants without nematode inoculation were used as the control. Two months after inoculation, all of the inoculated roots had galling symptoms similar to those observed in the field, and 100% of root system tissues had galls. The root gall index was ~6 according to a 0 to 10 RKN damage rating scale (Poudyal et al. 2005). No symptoms were found on the control plants. The nematodes were reisolated from root tissue and identified. M. incognita has a broad host range in many species of economic importance including Salvia miltiorrhiza (Wen et al. 2023), Ipomoea batatas (Maleita et al. 2022), and Zea mays (López-Robles et al. 2013). So far, M. incognita has been reported in D. alata and D. rotundata in Africa (Onkendi et al. 2014). To our best knowledge, this is the first record of M. incognita on D. opposita in Henan Province, China. With the increased planting area of D. opposita in China, root-knot nematodes are becoming more serious and reducing tuber production, with yield losses more than 60%. This identification is a preliminary step in developing effective disease management schemes. Declaration of interest The authors declare no conflict of interest. Funding This work was financially supported by the Key Scientific Research Projects of Higher Education Institutions of Henan Province (21A180013), China Agriculture Research System (CARS-21), The Zhongyuan high level talents special support plan-Science and Technology Innovation Leading Talents (224200510011) and Science and Technology Research Project of Henan Province (222102310211). References López-Robles, J., et al. 2013. Plant Dis. 97:694. https://doi.org/10.1094/PDIS-07-12-0674-PDN. Maleita, C., et al. 2022. Plant Dis. 106:2536. https://doi.org/10.1094/PDIS-12-21-2680-PDN. Meng, Q. P., et al. 2004. Acta Phytopathol. Sinica 34:204. https://doi.org/10.13926/j.cnki.apps.2004.03.003. Onkendi, E. M., et al. 2014. Plant Pathol. 63:727. https://doi.org/10.1111/ppa.12202. Poudyal, D. S., et al. 2005. Australas. Plant Pathol. 34:181. https://doi.org/10.1071/AP05011. Randig, O., et al. 2002. Genome 45:862. https://doi.org/10.1139/g02-054. Wen, Y., et al. 2023. Plant Dis. Accepted. https://doi.org/10.1094/PDIS-05-22-0997-PDN. Zijlstra, C., et al. 2000. Nematology 2:847. https://doi.org/10.1163/156854100750112798.

Molecular cloning and development of RAPD-SCAR markers for the selection of thermo-tolerant line of tropical tasar silkworm

Aim: To develop random amplified polymorphic DNA (RAPD) -sequence-characterized amplified region (SCAR) markers for selecting thermo-tolerant line of tropical tasar silkworm. Methodology: In the environmental chamber, Daba BV cocoons of A. mylitta were exposed to high temperature at 46°C/4 hr for 3 days. After the emergence, genomic DNA was extracted from thermo-tolerant moths and thermo-susceptible pupae. The genomic DNA was amplified using 30 RAPD random decamer primers. The improved RAPD fragments that can differentiate thermo-tolerant and susceptible line of A. mylitta were eluted, cloned in pJET1.2 vector and sequenced. SCAR markers were developed based on the sequence and validated in the subsequent generations. Results: Among 30 RAPD primers, OPK04, OPAJ15 and OPA17 generated polymorphic bands to differentiate thermo-tolerant and susceptible line of A. mylitta. These polymorphic bands were eluted, cloned and sequenced. Sequencing of three cloned fragments revealed that clone PB1 comprised of 1412 bp, clone PB2 comprised of 704 bp and clone PB3 comprised of 931 bp. Sequence specific stable multiplex SCAR markers TT-PB1, TT-PB2 and TT-PB3 were designed and synthesized. PCR amplification was performed using DNA templates of 10 thermo-tolerant and 10 thermo-susceptible samples. SCAR marker TT-PB1 was observed to be more specific to thermo-tolerant line of tropical tasar silkworm and validation with 25 samples each in next generation also supported the specificity of TT-PB1. Interpretation: Among three SCAR markers, TT-PB1 showed more specificity for selecting the thermo-tolerant line of A. mylitta. Therefore, the study provides an effective and precise PCR-based molecular marker system for selecting thermo-tolerant lines in tropical tasar silkworm to overcome seed crop loss due to high temperature stress in tasar rearing hotter zones. Key words: Antheraea mylitta, cloning, RAPD, SCAR m­­­arker, Thermo-tolerance