Genetic variations among the isolates of Bipolaris Maydis based on phenotypic and molecular markers

Maydis leaf blight, caused by Bipolaris maydis, is an important disease of maize crop in Khyber Pakhtunkhwa (KP) Pakistan. Fifteen isolates of the pathogen, collected across KP, were studied for variability based on phenotypic and molecular markers. Significant variability among the isolates was observed when assessed using phenotypic traits such as radial growth, spore concentration, fungicide sensitivity and virulence. The isolates were classified into six culture groups based on colour, texture and margins of the colony. Conidial morphology was also variable. These were either straight or slightly curved and light to dark brown in colour. Fungicide test showed significant variation in the degree of sensitivity against Carbendazim. Isolate Bm8 exhibited maximum radial growth on carbendazim spiked plates. Conversely, isolate Bm15 showed the lowest radial growth. Variations in virulence pattern of the isolates were evident when a susceptible maize variety Azam was inoculated with spores of B. maydis. Genetic variability amongst the isolates was also estimated by RAPD as well as sequencing of ITS region. The RAPD dendrogram grouped all the isolates into two major clusters. Average genetic distance ranged from 0.6% to 100%, indicating a diverse genetic gap among the isolates. Maximum genetic distance was found between isolates Bm9 and Bm10 as well as Bm2 and Bm8. Conversely, isolates Bm13 and Bm15 were at minimum genetic distance. Phylogenetic dendrogram based on sequencing of ITS region grouped all the isolates into a single major cluster. The clusters in both the dendrogram neither correlate to the geographical distribution nor to the morphological characteristics.

Isolation of Beauveria Strains and Their Potential as Control Agents for Lema bilineata Germar (Coleoptera: Chrysomelidae)

Lema bilineata Germar (Coleoptera: Chrysomelidae) was recently reported to damage Physalis peruviana crops in Brazil. Given the potential for inflicting damage on other Solanaceae species and the lack of alternatives for controlling this pest, we assessed the pathogenicity of 15 Beauveria isolates against L. bilineata adults in vitro. In addition, three of these isolates were tested for their ovicidal effect against L. bilineata eggs. Fungal strains were isolated from mummified corpses of L. bilineata collected in a non-commercial field in Paraná, Brazil. The isolates were identified as Beauveria bassiana using molecular markers. Lema bilineata adults were susceptible to conidial suspensions of all these isolates at a concentration of 108 conidia mL−1. Deaths caused by fungal extrusion were confirmed. Three strains were found to be more virulent against L. bilineata adults and showed ovicidal effects. This is the first study on entomopathogenic fungi isolated from dead insects collected from P. peruviana crops and tested against L. bilineata carried out in Brazil. The results obtained in the laboratory indicate the high potential of the use of three B. bassiana strains against L. bilineata as a biocontrol agent.

Identification of Parental Lines and Hybrid using Molecular Techniques in CGMS based Chilli (Capsicum annuum L.) Hybrid UARChH42 (JCH42)

Background: Molecular markers are the landmarks on DNA that identifies a particular sequence of base pairs coding for a character. SCAR (Sequenced Characterized Amplified Region) markers are proving to be more effective in identification of genotypes as they are PCR based co-dominant markers. In view of plant variety registration under PPV and FRA, 2001 molecular characterization/ identification of the variety is essential to ascertain the trueness of the variety, hence present studies have been planned and executed. Methods: In present investigation CGMS based chilli hybrid UARChH42 and its parental lines were identified using molecular techniques. A line, B line, R line and hybrid seedlings were used for DNA extraction and characterized on the basis of polymorphism with respect to sterility or fertility by using SCAR markers. Result: P1 and P2 and coxII-SCAR which are sterility specific markers could amplify A line and hybrid showing a definite band. CRF-SCAR 870 is a fertility specific marker amplified R line and hybrid. Also CMS-SCAR 130 and CMS-SCAR 130/140 were able to identified A line, B line, R line and hybrid. Two of the markers viz. orf-456-SCAR atp6-SCAR and were not able to specify any case of parental lines or hybrid identification. Hybridity of UARChH42 (JCH42) chilli hybrid was determined by any similarity in the banding pattern with any of its parent. This study would help in the fulfillment of the requirements of protection of plant varieties and farmers’ rights authority (PPVFRA), New Delhi for registration purpose.

Dietary Exposure to Flame Retardant Tris (2-Butoxyethyl) Phosphate Altered Neurobehavior and Neuroinflammatory Responses in a Mouse Model of Allergic Asthma

Tris (2-butoxyethyl) phosphate (TBEP) is an organophosphate flame retardant and used as a plasticizer in various household products such as plastics, floor polish, varnish, textiles, furniture, and electronic equipment. However, little is known about the effects of TBEP on the brain and behavior. We aimed to examine the effects of dietary exposure of TBEP on memory functions, their-related genes, and inflammatory molecular markers in the brain of allergic asthmatic mouse models. C3H/HeJSlc male mice were given diet containing TBEP (0.02 (TBEP-L), 0.2 (TBEP-M), or 2 (TBEP-H) μg/kg/day) and ovalbumin (OVA) intratracheally every other week from 5 to 11 weeks old. A novel object recognition test was conducted in each mouse at 11 weeks old. The hippocampi were collected to detect neurological, glia, and immunological molecular markers using the real-time RT-PCR method and immunohistochemical analyses. Mast cells and microglia were examined by toluidine blue staining and ionized calcium-binding adapter molecule (Iba)-1 immunoreactivity, respectively. Impaired discrimination ability was observed in TBEP-H-exposed mice with or without allergen. The mRNA expression levels of N-methyl-D aspartate receptor subunits Nr1 and Nr2b, inflammatory molecular markers tumor necrosis factor-α oxidative stress marker heme oxygenase 1, microglia marker Iba1, and astrocyte marker glial fibrillary acidic protein were significantly increased in TBEP-H-exposed mice with or without allergen. Microglia and mast cells activation were remarkable in TBEP-H-exposed allergic asthmatic mice. Our results indicate that chronic exposure to TBEP with or without allergen impaired object recognition ability accompanied with alteration of molecular expression of neuronal and glial markers and inflammatory markers in the hippocampus of mice. Neuron-glia-mast cells interaction may play a role in TBEP-induced neurobehavioral toxicity.

Evaluation of resistant genotypes and their characterization using molecular markers linked for powdery mildew resistance in cucumber (Cucumis sativus L.)

Powdery mildew (PM) is one of the most severe fungal diseases of cucumber that limits its production worldwide. In this study, 140 genotypes of cucumber were screened for disease resistance under field and artificial conditions, and then validated with eight known SSR markers linked to PM resistance. Among these genotypes, genotype GS140 was found resistant (R), whereas GS148, GS16 and GS70 were moderately resistant, and GS169 was found to be tolerant. Of all the eight markers, only C31, C80, C162, SSR16472 and SSR16881 amplified the reported linked allele. The 127 bp allele of SSR16881 was found to be associated with the lowest disease severity of 37.65%. The associated markers could further be verified for their usability using linkage studies and the contrast genotypes in the present study could serve as a tool for selection in future marker-assisted selection breeding strategies for PM resistance.

Species discrimination of novel chloroplast DNA barcodes and their application for identification of Panax (Aralioideae, Araliaceae)

Certain species within the genus Panax L. (Araliaceae) contain pharmacological precious ginsenosides, also known as ginseng saponins. Species containing these compounds are of high commercial value and are thus of particular urgency for conservation. However, within this genus, identifying the particular species that contain these compounds by morphological means is challenging. DNA barcoding is one method that is considered promising for species level identification. However, in an evolutionarily complex genus such as Panax, commonly used DNA barcodes such as nrITS, matK, psbA-trnH, rbcL do not provide species-level resolution. A recent in silico study proposed a set of novel chloroplast markers, trnQ-rps16, trnS-trnG, petB, and trnE-trnT for species level identification within Panax. In the current study, the discriminatory efficiency of these molecular markers is assessed and validated using 91 reference barcoding sequences and 38 complete chloroplast genomes for seven species, one unidentified species and one sub-species of Panax, and two outgroup species of Aralia L. along with empirical data of Panax taxa present in Vietnam via both distance-based and tree-based methods. The obtained results show that trnQ-rps16 can classify with species level resolution every clade tested here, including the highly valuable Panax vietnamensis Ha et Grushv. We thus propose that this molecular marker to be used for identification of the species within Panax to support both its conservation and commercial trade.

Development of PCR-Based Race-Specific Markers for Differentiation of Indian Fusarium oxysporum f. sp. cubense, the Causal Agent of Fusarium Wilt in Banana

Fusarium wilt caused by Fusarium oxysporum f. sp. cubense (Foc), is the most lethal soil-borne fungal pathogen infecting bananas. Foc race 1 (R1) and 4 (R4) are the two most predominant races affecting the economically important Cavendish group of bananas in India. A total of seven vegetative compatibility groups (VCGs) from three pathogenic races were isolated during our field survey and were found to be highly virulent towards cv. Grande Naine. According to comparative genome analyses, these Indian Foc VCGs were diverse in genomic organization and effector gene profiles. As a result, false-positive results were obtained with currently available molecular markers. In this context, the study has been initiated to develop PCR-based molecular markers for the unambiguous identification of Indian Foc R1 and R4 isolates. Whole-genome sequences of Foc R1 (GCA_011316005.3), Foc TR4 (GCA_014282265.3), and Foc STR4 (GCA_016802205.1), as well as the reference genomes of Foc (ASM799451v1) and F. oxysporum f. sp. lycopersici (Fol; ASM14995v2), were aligned to identify unique variable regions among the Foc races. Using putative chromosome and predicted gene comparison, race-specific unique Foc virulence genes were identified. The putative lineage-specific identified genes encoding products secreted in xylem (SIX) that may be necessary for disease development in the banana. An in silico analysis was performed and primers were designed from a region where sequences were dissimilar with other races to develop a specific marker for Foc R1, R4, TR4, and STR4. These race-specific markers allowed target amplification in the characterized highly virulent Foc isolates, and did not show any cross-amplification to any other Foc races, VCGs or banana pathogens, Fusarium species, and non-pathogenic Fusarium oxysporum isolates. The study demonstrated that the molecular markers developed for all the three Foc races of India could detect the pathogen in planta and up to 0.025 pg µL−1 DNA levels. Thus, the markers developed in this study are novel and could potentially be useful for the accurate diagnosis and detection of the Indian Foc races which are important for the effective management of the disease.