First report of Phytopythium vexans causing gummosis and root rot of Khasi Mandarin (Citrus reticulata Blanco) in north eastern states of India.

Khasi mandarin (Citrus reticulata Blanco) is the most economically important crop among the citrus growing region in the north-eastern India (Singh et al. 2016). An extensive survey was conducted to identify the causal agent of citrus root rot and gummosis in north eastern states (Meghalaya, Tripura, Manipur, Arunachal Pradesh, Sikkim, Nagaland and Assam) of India during October 2021-23. The gummosis disease incidence ranged from 5 to 95 % in 10 to 25 years old Khasi mandarin plants showing relatively more chronic symptoms on mature trees. Yellowing and dropping of leaves, twigs die back, gum oozing from infected bark and loss of feeder roots were the typical symptoms of the disease. Infected bark tissue and young lemon leaf baits in rhizosphere soil were plated on corn meal agar medium supplemented with pimaricin (10 µg/ml), ampicillin (250 µg/ml), rifamycin (10 µg/ml) and 300µg/ml carbendazim and incubated at 26℃. Fifty isolates were purified and maintained on Carrot agar medium. These isolates showed similar cultural and morphological characteristics. Two representative isolates from Arunachal Pradesh (AP21 and AP26) were selected for further experiments and deposited to Indian Type culture collection (ITCC), New Delhi with accession no. 9156 and 9157 respectively. The colonies were fast growing, showing rosette pattern along with whitish blooming mycelium appearance with no visual sporulation at the surface. The hyphae were coenocytic with initially right-angled branching. Sporangia were globose or sub globose and papillated. Oogonia were smooth and globose (16.29-21.09 µm) in diameter. Antheridia were irregular, cylindrical and broadly attached to oogonia. Empty sporangia were also observed. Multilocus phylogenetic analysis using internal transcribed spacer region (Das et al. 2011), ß tubulin (Blair et al. 2008) and Cytochrome oxidase II gene (Noireung et al. 2020) showed that these isolates formed a stable clade with Phytopythium vexans (CBS119.80) sequence retrieved from NCBI database. BLAST analysis showed that ITS sequence of AP21 (OQ372986) and AP26 (OQ381083) had >99 % identity with P. vexans isolate NS-3 (ON533631). Further, BLAST analysis of ß tubulin (AP21 OQ446053, AP26 OR405377) and Cox II gene (AP21 OQ473414, AP26 OR552422) sequences showed that our Indian isolates showed >99 % similarity with P. vexans voucher strain CBS119.80. To fulfil Koch's postulates, Khasi mandarin (Citrus reticulata) seedlings were inoculated by adding 100 ml zoospore suspension of P. vexans (1x105 spores/ml) in sterilized soil (Thao et al. 2020). The experiment was carried out in triplicate. Yellowing of leaves and leaf drop were observed 7 days post inoculation while 30 days post inoculation, treated plants started showing symptoms of root rot, including mild root decay. No symptoms were observed in control treatment. The pathogen was reisolated from symptomatic roots and confirmed through colony and sporangium morphology. Recently, it was reported that P. vexans is associated with apple and pear decline in the Saiss plain of Morocco (Jabiri et al. 2021), root rot on mandarin in Thailand (Noireung et al. 2020) and on Durian in Vietnam (Thao et al. 2020). As per our knowledge, this is the first report of P. vexans causing root rot and gummosis in Khasi mandarin from north eastern states of India. This finding is significantly important for the development of a successful disease management strategy in India.

3Bs of CRISPR-Cas mediated genome editing in plants: exploring the basics, bioinformatics and biosafety landscape.

The recent thrust in research has projected the type II clustered regularly interspaced short palindromic repeats and associated protein 9 (CRISPR-Cas9) system as an avant-garde plant genome editing tool. It facilitates the induction of site-specific double-stranded DNA cleavage by the RNA-guided DNA endonuclease (RGEN), Cas9. Elimination, addition, or alteration of sections in DNA sequence besides the creation of a knockout genotype (CRISPRko) is aided by the CRISPR-Cas9 system in its wild form (wtCas9). The inactivation of the nuclease domain generates a dead Cas9 (dCas9), which is capable of targeting genomic DNA without scissoring it. The dCas9 system can be engineered by fusing it with different effectors to facilitate transcriptional activation (CRISPRa) and transcriptional interference (CRISPRi). CRISPR-Cas thus holds tremendous prospects as a genome-manipulating stratagem for a wide gamut of crops. In this article, we present a brief on the fundamentals and the general workflow of the CRISPR-Cas system followed by an overview of the prospects of bioinformatics in propelling CRISPR-Cas research with a special thrust on the available databases and algorithms/web-accessible applications that have aided in increasing the usage and efficiency of editing. The article also provides an update on the current regulatory landscape in different countries on the CRISPR-Cas edited plants to emphasize the far-reaching impact of the genomic editing technology. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01397-3.

Transcriptome Analysis of Wheat-Tilletia indica Interaction Provides Defense and Pathogenesis-Related Genes.

Karnal bunt (Tilletia indica Mitra) is an internationally quarantined disease of wheat. Until now, very little information has been available on the molecular basis of resistance and pathogenicity of T. indica. To investigate the molecular basis of host−pathogen interaction, the transcriptome of T. indica inoculated resistant (HD29) and susceptible (WH542) genotypes of wheat were analyzed. Approximately 58 million reads were generated using RNA sequencing by the Illumina NextSeq500 platform. These sequence reads were aligned to a reference genome of wheat to compare the expression level of genes in resistant and susceptible genotypes. The high-quality reads were deposited in the NCBI SRA database (SRP159223). More than 80,000 genes were expressed in both the resistant and susceptible wheat genotypes. Of these, 76,088 were commonly expressed genes, including 3184 significantly upregulated and 1778 downregulated genes. Four thousand one hundred thirteen and 5604 genes were exclusively expressed in susceptible and resistant genotypes, respectively. Based on the significance, 503 genes were upregulated and 387 genes were downregulated. Using gene ontology, the majority of coding sequences were associated with response to stimuli, stress, carbohydrate metabolism, developmental process, and catalytic activity. Highly differentially expressed genes (integral component of membrane, exonuclease activity, nucleic acid binding, DNA binding, metal ion binding) were validated in resistant and susceptible genotypes using qPCR analysis and similar expression levels were found in RNA-Seq. Apart from the wheat, the mapping of T. indica was 7.07% and 7.63% of resistant and susceptible hosts, respectively, upon infection, which revealed significant pathogenesis-related genes. This first study provided in-depth information and new insights into wheat−T. indica interaction for managing Karnal bunt disease of wheat.

Detection of Mesta yellow vein mosaic virus (MeYVMV) in field samples by a loop-mediated isothermal amplification reaction.

A loop-mediated isothermal amplification (LAMP) assay was optimized for the detection of Mesta yellow vein mosaic virus (MeYVMV) in diseased plants of mesta (Hibiscus sabdariffa L.& H. cannabinus L.). The LAMP assay was optimized using a set of six primers targeting the MeYVMV genome and could be completed in 30-60 min at 63 °C. The LAMP amplification results were visualized by adding 1 µl of hydroxy naphthol blue (HNB) dye in a 25 µl LAMP reaction mixture prior to amplification as well as by electrophoresis. The LAMP assay, which detected MeYVMV in a 10-5-fold diluted total DNA, was more sensitive than the PCR assay (10-4-fold dilution). The optimized LAMP assay was able to detect MeYVMV in different parts of the kenaf and roselle plants. Similarly, the optimized PCR assay was also capable of detecting MeYVMV in all the different parts of the kenaf plant but failed to detect the virus in the stem and flower buds of the roselle plant. Validation of the LAMP and LAMP with HNB dye assays revealed that the optimized reactions can be used successfully for the in-situ detection of MeYVMV in field samples and in virus quarantine programs. This is the first report of the detection of the begomovirus species, MeYVMV, in the mucilaginous plant species, kenaf and roselle, using a LAMP assay.

Impact of post-anthesis rainfall, fungicide and harvesting time on the concentration of deoxynivalenol and zearalenone in wheat.

Field experiments were conducted to identify the impact of post-anthesis rainfall on the concentration of deoxynivalenol (DON) and zearalenone (ZON) in harvested wheat grain. Winter wheat plots were inoculated with Fusarium graminearum at stem extension (GS31) and prothioconazole was applied at mid-anthesis (GS65) to split plots and plots were subsequently mist irrigated for 5 days. Plots were either covered by polytunnels, irrigated by sprinklers or left as non-irrigated uncovered control plots after medium-milk (GS75). Plots were harvested either when ripe (GS92; early harvest) or three weeks later (late harvest). Fusarium head blight (FHB) was assessed each week from inoculation. At harvest, yield and grain quality was measured and grains were analysed for DON and ZON. Differences in rainfall resulted in contrasting disease pressure in the two experiments, with low FHB in the first experiment and high FHB in the second. Difference in FHB resulted in large differences in grain yield, quality and mycotoxin content. DON concentration was significantly (P < 0.05) higher in irrigated compared to covered and control plots in the first experiment, whereas in the second experiment, DON was significantly (P < 0.05) higher in the covered plots compared to the control and irrigated plots. ZON concentration was significantly (P < 0.05) higher in irrigated plots in both experiments. Later harvesting resulted in an approximate fivefold increase in ZON in the first experiment, but was not significantly different in the second experiment. Prothioconazole significantly (P < 0.05) reduced DON in both experiments, but gave inconsistent reductions to ZON. This is the first report to show that the post-anthesis rainfall can significantly increase ZON in wheat, which can increase further with a delayed harvest but may be significantly reduced with the application of prothioconazole. Importantly, in the absence of moisture late season, ZON remains at very low concentrations even when wheat is severely affected by FHB.

Molecular characterization of Chaetomium species using URP-PCR

Chaetomium spp. are common colonizers of soil and cellulose-containing substrates. Seventeen isolates of Chaetomium spp., which included 15 isolates of C. globosum and one each of C. reflexum and C. perlucidum, were genetically characterized with universal rice primers (URP - primers derived from DNA repeat sequences in the rice genome) using polymerase chain reaction (URP-PCR). Out of the 12 URP's used in the study, nine primers were effective in producing polymorphic fingerprint patterns from DNA of Chaetomium spp. Analysis of the entire fingerprint profile using the unweighted pair-group method with arithmetic averages (UPGMA) clearly differentiated C. globosum isolates from C. perlucidum and C. reflexum. One of the primers, URP-2R, produced a uniform DNA band of 1.9 kb in all the isolates of C. globosum but not in C. perlucidum and C. reflexum, which can be used as molecular marker to differentiate C. globosum from other species. Our results indicate that URP's are sensitive and give reproducible results for assaying the genetic variability in Chaetomium spp.