First report of Colletotrichum orbiculare causing anthracnose of pointed gourd in India.

Pointed gourd (Trichosanthes dioica Roxb.), of the Cucurbitaceae family, is widely cultivated as a vegetable in many countries such as Bangladesh, India, Pakistan, Myanmar, Nepal and Sri Lanka. Over 800,000 metric tons of pointed gourds are produced annually in India, where cultivation is estimated to occupy over 33,000 hectares of land (MoA & FW, Government of India). In summer 2018, significant losses (approximately 15-20%) occurred in the sub-Himalayan region in West Bengal state of India (21.14-21.30° N, 78.82-79.02°E) due to a disease with typical anthracnose-like symptoms on the fruits. Light yellowish, small sized round to irregular spots were also apparent on the leaves. These spots gradually increased in size and turned into light brown and were surrounded by yellow halo. The lesions on the fruits were circular, yellow-brown, necrotic and sunken. A survey of four fields (1.5 ha) was conducted and a disease incidence of 30-40% was observed. Necrotic tissues from fruit as well as leaves were cut into approximately 5 mm2, surface sterilized with 0.1% HgCl2, plated in potato dextrose agar and incubated at 28ºC for 7 days in the dark. A total of 50 morphologically similar colonies were obtained from 20 sampled fruits and 10 sampled leaves. Fungal colonies were initially white, becoming gray as the cultures aged on PDA. The cultures developed black acervuli around the center of the colony. Setae were brown in colour, 1-5 septate, 40-100 µm long. Conidia were also observed through light and scanning electron microscopy and exhibit as (4-6 ×13-19 µm) hyaline, aseptate, cylindrical to oblong, with one end round and other truncate. The morphological characteristics were found similar to Colletotrichum orbiculare Damm, P.F. Cannon & Crous as reported by Damm et al. (2013). Ten isolates were obtained by transferring hyphal tips to new PDA plates and incubating under the same conditions. To confirm the identity of the pathogen, genomic DNA was extracted from five pure isolates (PG-Pha, PG-Pha-2, PG-Pha-3, PG-Pha-4, PG-Pha-5) with the cetyltrimethylammonium bromide (CTAB). Further, the ITS1-5.8S-ITS2 region, D1/D2 region of the 28S rRNA large subunit (LSU), Actin (ACT) gene and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene were amplified using specific primers, ITS1/ITS4 (White et al. 1990), NL1/NL4, ACT1/ACT2 and GDF1/GDR1 respectively and PCR conditions described in Damm et al. (2012). A GenBank BLAST search showed 99-100% identity to the Colletotrichum orbiculare (Acc. Nos. KP898988 for ITS1-5.8S-ITS2, Z18997 for 28S rRNA, AB778553 for ACT gene, and KF178482 for GAPDH). All obtained sequences were submitted to the GenBank (Acc. Nos. MN006616, OP811046-OP811049, [ITS1-5.8-ITS2], MN006684, PP391616-PP391619 [28S rRNA], MN168524, PP400822-PP400825 [ACT gene], OP627091, PP400826-PP400829 [GAPDH]). For phylogenetic analysis, MEGA version 11 (Tamura et al. 2021) was used to construct a maximum likelihood tree with 1000 bootstrap replicates, based on a concatenation alignment of three gene sequences (ITS, Actin and GAPDH) of the all the five C. orbiculare isolates as well as sequences of other Colletotrichum species obtained from GenBank. The cluster analysis revealed that, isolate PG-Pha form a cluster with other C. orbiculare isolates. Pathogenicity tests were conducted to confirm Koch's postulates. Pathogenicity tests were performed in mature fruits by inoculating them (n=8) with 10 µl of a 1×106 conidia/ml suspension at needle puncture wound sites. In control set up sterile distilled water was pipetted on fruits. Fruits were placed on sterile trays covered with glasses and incubated at humid chambers at 28±2ºC with 12 h of light. Healthy one-month old potted pointed gourd plants (n=15) were sprayed with conidial suspension until run-off. A set of 15 plants were sprayed with sterile distilled water and maintained as control. The plants were kept in a greenhouse at 25ºC, >75% relative humidity, and a 16/8 h day/night cycle for 15 days. Sterile distilled water was sprayed on the plants at one day interval to maintain the humidity. Inoculated fruits started showing yellowing symptoms one day post inoculation and gradually yellow-brown sunken spots became visible at the place of puncture, whereas control fruits remain symptomless even after 7 days of inoculation. Inoculated leaves showed disease symptoms similar to those observed in the field whereas leaves of control sets were symptomless even after 15 days. The pathogenicity test was repeated thrice under the same conditions mentioned before. C. orbiculare was successfully re-isolated from all the symptomatic tissues of leaves as well as fruits, completing Koch's postulates. Previously, the pathogen has been reported as an important anthracnose pathogen of Cucurbitaceae, especially of cucumber (Cucumis sativus), melons (Cucumis melo), watermelon (Citrullus lanatus), pumpkin (Cucurbita pepo) and squash (Cucurbita maxima) (Farr and Rossman 2020). To our knowledge, this is the first report of C. orbiculare causing anthracnose of pointed gourd. This disease represents a threat to producers in India and central Asia. Further research may contribute to the development of management strategies for this disease.

Phylogeny and synonymous codon usage pattern of Papaya ringspot virus coat protein gene in the sub-Himalayan region of north-east India.

Sub-Himalayan West Bengal is favorable for the production of several fruits and vegetables. Papaya is one of the common plants cultivated in the area. Most of the papaya plants of the area are susceptible to Papaya ringspot virus (PRSV). Coat protein genes of 6 PRSV isolates of the area were sequenced following RT-PCR. Phylogenetic study of the PRSV isolates showed about 80%-90% similarity with Cuban isolates. The codon usage pattern of our isolates was also analyzed, along with several other isolates. PRSV isolates of our study showed a preference for 8 putative optimal codons. Correspondence analysis of the genes of different isolates along the first 2 major axes were done, as the first 2 axes contributed more in shaping codon usage pattern. In the phylogenetic tree constructed by the neighbour-joining method, our isolates clustered together with the east Indian, north Indian, and Bangladeshi isolates. The diversity and codon usage pattern of the PRSV isolates of different regions were studied, and it was observed that the codon usage pattern of PRSV isolates is probably influenced by translational selection along with mutational bias.

NSCLC: from tumorigenesis, immune checkpoint misuse to current and future targeted therapy.

Non-small cell lung cancer (NSCLC) is largely promoted by a multistep tumorigenesis process involving various genetic and epigenetic alterations, which essentially contribute to the high incidence of mortality among patients with NSCLC. Clinical observations revealed that NSCLC also co-opts a multifaceted immune checkpoint dysregulation as an important driving factor in NSCLC progression and development. For example, a deregulated PI3K/AKT/mTOR pathway has been noticed in 50-70% of NSCLC cases, primarily modulated by mutations in key oncogenes such as ALK, EGFR, KRAS, and others. Additionally, genetic association studies containing patient-specific factors and local reimbursement criteria expose/reveal mutations in EGFR/ALK/ROS/BRAF/KRAS/PD-L1 proteins to determine the suitability of available immunotherapy or tyrosine kinase inhibitor therapy. Thus, the expression of such checkpoints on tumors and immune cells is pivotal in understanding the therapeutic efficacy and has been extensively studied for NSCLC treatments. Therefore, this review summarizes current knowledge in NSCLC tumorigenesis, focusing on its genetic and epigenetic intricacies, immune checkpoint dysregulation, and the evolving landscape of targeted therapies. In the context of current and future therapies, we emphasize the significance of antibodies targeting PD-1/PD-L1 and CTLA-4 interactions as the primary therapeutic strategy for immune system reactivation in NSCLC. Other approaches involving the promising potential of nanobodies, probodies, affibodies, and DARPINs targeting immune checkpoints are also described; these are under active research or clinical trials to mediate immune regulation and reduce cancer progression. This comprehensive review underscores the multifaceted nature, current state and future directions of NSCLC research and treatment.

Topical Spray of dsRNA Induces Mortality and Inhibits Chilli Leaf Curl Virus Transmission by Bemisia tabaci Asia II 1.

Chilli leaf curl virus (ChiLCV; genus: Begomovirus), transmitted by Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) in a persistent-circulative manner, is a major constraint in chilli production. The present study demonstrates for the first time that a topical spray of naked double-stranded RNA (dsRNA) on chilli plants causes mortality and inability to acquire and transmit ChiLCV in B. tabaci. dsRNA targeting heat shock protein 70 (hsp70) and fasciclin 2 (fas2) of B. tabaci Asia II 1 was first assessed under controlled conditions through oral delivery. Hsp70 and fas2 dsRNA resulted in up to 82.22% and 72% mortality of B. tabaci and around 12.4- and 8.5-fold decreases in mRNA levels, respectively, 24 h post-ingestion. ChiLCV copies in hsp70 dsRNA-fed B. tabaci steadily decreased with an increase in dsRNA concentration and were undetectable at a higher concentration of dsRNA. However, ChiLCV copies significantly increased in fas2 dsRNA-fed B. tabaci. Transmission of ChiLCV by B. tabaci was completely inhibited post-24 h feeding on hsp70 dsRNA at 3 µg/mL. Naked hsp70 dsRNA was topically sprayed on ChiLCV-infected chilli plants like an insecticide. 67.77% mortality of B. tabaci, 4.6-fold downregulation of hsp70 mRNA, and 1.34 × 1015-fold decreased ChiLCV copies in B. tabaci were recorded when adults were exposed to the dsRNA-treated plants under semi-field conditions. Foliar application of naked dsRNA reduced the ChiLCV transmission by 75% without any visible symptoms in the inoculated plants. A total of 2 consecutive sprays of dsRNA provided significant protection to B. tabaci for up to 20 days under semi-field conditions.

Transcriptomic Changes of Bemisia tabaci Asia II 1 Induced by Chilli Leaf Curl Virus Trigger Infection and Circulation in Its Vector.

Bemisia tabaci (Hemiptera: Aleyrodidae) is a highly efficient vector in the spread of chilli leaf curl virus (ChiLCV, Begomovirus) which is a major constraint in the production of chilli in South Asia. Transcriptome analysis of B. tabaci post-6 h acquisition of ChiLCV showed differential expression of 80 (29 upregulated and 51 downregulated) genes. The maximum number of DEGs are categorized under the biological processes category followed by cellular components and molecular functions. KEGG analysis of DEGs showed that the genes are involved in the functions like metabolism, signaling pathways, cellular processes, and organismal systems. The expression of highly expressed 20 genes post-ChiLCV acquisition was validated in RT-qPCR. DEGs such as cytosolic carboxypeptidase 3, dual-specificity protein phosphatase 10, 15, dynein axonemal heavy chain 17, fasciclin 2, inhibin beta chain, replication factor A protein 1, and Tob1 were found enriched and favored the virus infection and circulation in B. tabaci. The present study provides an improved understanding of the networks of molecular interactions between B. tabaci and ChiLCV. The candidate genes of B. tabaci involved in ChiLCV transmission would be novel targets for the management of the B. tabaci-begomovirus complex.

How many begomovirus copies are acquired and inoculated by its vector, whitefly (Bemisia tabaci) during feeding?

Begomoviruses are transmitted by whitefly (Bemisia tabaci Gennadius, Hemiptera: Aleyrodidae) in a persistent-circulative way. Once B. tabaci becomes viruliferous, it remains so throughout its life span. Not much is known about the copies of begomoviruses ingested and/or released by B. tabaci during the process of feeding. The present study reports the absolute quantification of two different begomoviruses viz. tomato leaf curl New Delhi virus (ToLCNDV, bipartite) and chilli leaf curl virus (ChiLCV, monopartite) at different exposure of active acquisition and inoculation feeding using a detached leaf assay. A million copies of both the begomoviruses were acquired by a single B. tabaci with only 5 min of active feeding and virus copy number increased in a logarithmic model with feeding exposure. Whereas, a single B. tabaci could inoculate 8.21E+09 and 4.19E+11 copies of ToLCNDV and ChiLCV, respectively in detached leaves by 5 min of active feeding. Virus copies in inoculated leaves increased with an increase in feeding duration. Comparative dynamics of these two begomoviruses indicated that B. tabaci adult acquired around 14-fold higher copies of ChiLCV than ToLCNDV 24 hrs post feeding. Whereas, the rate of inoculation of ToLCNDV by individual B. tabaci was significantly higher than ChiLCV. The study provides a better understanding of begomovirus acquisition and inoculation dynamics by individual B. tabaci and would facilitate research on virus-vector epidemiology and screening host resistance.

Codon Usage Bias Analysis of Citrus tristeza Virus: Higher Codon Adaptation to Citrus reticulata Host.

Citrus tristeza virus (CTV), a member of the aphid-transmitted closterovirus group, is the causal agent of the notorious tristeza disease in several citrus species worldwide. The codon usage patterns of viruses reflect the evolutionary changes for optimization of their survival and adaptation in their fitness to the external environment and the hosts. The codon usage adaptation of CTV to specific citrus hosts remains to be studied; thus, its role in CTV evolution is not clearly comprehended. Therefore, to better explain the host⁻virus interaction and evolutionary history of CTV, the codon usage patterns of the coat protein (CP) genes of 122 CTV isolates originating from three economically important citrus hosts (55 isolate from Citrus sinensis, 38 from C. reticulata, and 29 from C. aurantifolia) were studied using several codon usage indices and multivariate statistical methods. The present study shows that CTV displays low codon usage bias (CUB) and higher genomic stability. Neutrality plot and relative synonymous codon usage analyses revealed that the overall influence of natural selection was more profound than that of mutation pressure in shaping the CUB of CTV. The contribution of high-frequency codon analysis and codon adaptation index value show that CTV has host-specific codon usage patterns, resulting in higheradaptability of CTV isolates originating from C. reticulata (Cr-CTV), and low adaptability in the isolates originating from C. aurantifolia (Ca-CTV) and C. sinensis (Cs-CTV). The combination of codon analysis of CTV with citrus genealogy suggests that CTV evolved in C. reticulata or other Citrus progenitors. The outcome of the study enhances the understanding of the factors involved in viral adaptation, evolution, and fitness toward their hosts. This information will definitely help devise better management strategies of CTV.