Development of Reverse Transcription Recombinase Polymerase Amplification (RT-RPA): A Methodology for Quick Diagnosis of Potato Leafroll Viral Disease in Potato.

Potatoes are developed vegetatively from tubers, and therefore potato virus transmission is always a possibility. The potato leafroll virus (PLRV) is a highly devastating virus of the genus Polerovirus and family Luteoviridae and is regarded as the second-most destructive virus after Potato virus Y. Multiple species of aphids are responsible for the persistent and non-propagating transmission of PLRV. Due to intrinsic tuber damage (net necrosis), the yield and quality are drastically diminished. PLRV is mostly found in phloem cells and in extremely low amounts. Therefore, we have attempted to detect PLRV in both potato tuber and leaves using a highly sensitive, reliable and cheap method of one-step reverse transcription-recombinase polymerase amplification (RT-RPA). In this study, an isothermal amplification and detection approach was used for efficient results. Out of the three tested primer sets, one efficiently amplified a 153-bp product based on the coat protein gene. In the present study, there was no cross-reactivity with other potato viruses and the optimal amplification reaction time was thirty minutes. The products of RT-RPA were amplified at a temperature between 38 and 42 °C using a simple heating block/water bath. The present developed protocol of one-step RT-RPA was reported to be highly sensitive for both leaves and tuber tissues equally in comparison to the conventional reverse transcription-polymerase chain reaction (RT-PCR) method. By using template RNA extracted employing a cellular disc paper-based extraction procedure, the method was not only simplified but it detected the virus as effectively as purified total RNA. The simplified one-step RT-RPA test was proven to be successful by detecting PLRV in 129 samples of various potato cultivars (each consisting of leaves and tubers). According to our knowledge, this is the first report of a one-step RT-RPA performed using simple RNA extracted from cellular disc paper that is equally sensitive and specific for detecting PLRV in potatoes. In terms of versatility, durability and the freedom of a highly purified RNA template, the one-step RT-RPA assay exceeds the RT-PCR assay, making it an effective alternative for the certification of planting materials, breeding for virus resistance and disease monitoring.

Mechanistic insights on melatonin-mediated drought stress mitigation in plants.

Drought stress imposes a serious threat to crop productivity and nutritional security. Drought adaptation mechanisms involve complex regulatory network comprising of various sensory and signaling molecules. In this context, melatonin has emerged as a potential signaling molecule playing a crucial role in imparting stress tolerance in plants. Melatonin pretreatment regulates various plant physiological processes such as osmoregulation, germination, photosynthesis, senescence, primary/secondary metabolism, and hormonal cross-talk under water deficit conditions. Melatonin-mediated regulation of ascorbate-glutathione (AsA-GSH) cycle plays a crucial role to scavenge reactive oxygen species generated in the cells during drought. Here, in this review, the current knowledge on the role of melatonin to ameliorate adverse effects of drought by modulating morphological, physiological, and redox regulatory processes is discussed. The role of melatonin to improve water absorption capacity of roots by regulating aquaporin channels and hormonal cross-talk involved in drought stress mitigation are also discussed. Overall, melatonin is a versatile bio-molecule involved in growth promotion and yield enhancement under drought stress that makes it a suitable candidate for eco-friendly crop production to ensure food security.

Estragole, a potential attractant of the winged melon aphid Aphis gossypii.

BACKGROUND: Aphids are sap-sucking insect pests of economic importance. They exhibit polyphenism, producing two kinds of morphotypes; winged (alate) and wingless (aptera) morphs. While wingless morphs can be controlled by insecticides, winged morphs are a challenge for targeted control measures as they can fly. Although colored sticky traps are used to control and monitor winged aphids, only a small population is trapped, making sticky traps less effective in controlling aphids. Studies have shown that fragrant oils applied to sticky traps increased attraction of sap-sucking insects like whiteflies and thrips. Here, we tested selected essential oils for their attractiveness to winged Aphis gossypii in potato fields. RESULTS: In field assays, selected essential oils with yellow or colorless sticky traps attracted more winged A. gossypii than controls. The combination of yellow traps baited with essential oils attracted ~2-3-fold more winged A. gossypii than did colorless traps baited with essential oils. In a multi-cycle 2 year study, yellow sticky traps baited with basil oil consistently attracted more winged A. gossypii than yellow sticky traps baited with lavender, geranium or tea tree oils. In electrophysiological studies, winged A. gossypii's antennae responded consistently to estragole in basil oil. In olfactometer assays with estragole, winged A. gossypii spent significantly more time in the treatment arm of the olfactometer than in the control arm, validating estragole's attractiveness. Furthermore, yellow sticky traps baited with pure estragole, in potato fields, attracted similar number of winged A. gossypii as yellow sticky traps baited with basil oil. CONCLUSION: Our findings demonstrate the potential of using basil oil as a potential attractant to improve the efficacy of sticky traps in the monitoring and control of winged aphids. © 2023 Society of Chemical Industry.

Dynamic changes in virus-induced volatiles in cotton modulate the orientation and oviposition behavior of the whitefly Bemisia tabaci.

Manipulation of insect vector behavior by virus-induced plant volatiles is well known. But how the viral disease progression alters the plant volatiles and its effect on vector behavior remains less explored. Our studies tracked changes in volatile profile in progressive infection stages of cotton leaf curl virus (CLCuV) infected plants and their effect on B. tabaci behavior. Significant differences in virus titers were noticed between progressive infection stages showing distinct symptoms. Whiteflies initially settled on CLCuV infected plants, but their preference was shifted to healthy plants over time. GC-MS analysis revealed subtle quantitative/qualitative changes in volatile organic compounds (VOCs) between the healthy and selected CLCuV infection stages. VOCs such as hexanal, (E)-2-hexen-1-ol, (+)-α-pinene, (-)-ß-pinene, (Z)-3-hexen-1-ol, (+)-sylvestrene, and (1S,2E,6E, 10R)-3,7,11,11-tetramethylbicycloundeca-2,6-diene (Bicyclogermacrene) were associated with the infection stage showing upward curling of leaves; (E)-2-hexen-1-ol, ß-myrcene, ß-ocimene, and copaene were associated with the infection stage showing downward curling. Validation studies with eight synthetic VOCs indicated that γ-terpinene elicited attraction to B. tabaci (Olfactometric Preference Index (OPI) = 1.65), while ß-ocimene exhibited strong repellence (OPI = 0.64) and oviposition reduction (66.01%-92.55%). Our studies have demonstrated that progression of CLCuV disease in cotton was associated with dynamic changes in volatile profile which influences the behavioural responses of whitefly, B.tabaci. Results have shown that VOCs such as (+)-α-pinene, (-)-ß-pinene γ-Terpinene, α-guaiene; 4- hydroxy- 4 methyl-2- pentanone and ß-ocimene emitted from Begomovirus infected plants could be the driving force for early attraction and later repellence/oviposition deterrence of B. tabaci on virus-infected plants. The findings of this study offer scope for the management of whitefly, B. tabaci through semiochemicals.

Establishment of a one-step reverse transcription recombinase polymerase amplification assay for the detection of potato virus S.

Potato virus S (PVS) is a noteworthy threat to the propagation of healthy seed potatoes. Accurate and speedy detection is critical for effective PVS management. In the present study, an isothermal-based one-step reverse transcription-recombinase polymerase amplification (RT-RPA) approach was developed to detect PVS infection in potato leaves and tubers. A primer set based on the coat protein gene successfully amplified a 158 bp product out of three primer sets examined. The amplification reaction took less than 30 min to complete with no account of cross-reactivity with major potato viruses. Additionally, amplification of RT-RPA products was performed on the heating system and/or water bath at 38-42 °C. The results of sensitivity analysis revealed that one-step RT-RPA has shown 100 times higher sensitivity than routine RT-PCR for the detection of PVS in infected leaves. Furthermore, ten times higher sensitivity of RT-RPA was observed in infected tubers. The methodology was simplified further by the use of template RNA extracted using a cellular disc paper-based extraction method that detected the PVS more effectively than purified total RNA. PVS was detected in 175 samples (leaves and tubers each) of several potato varieties using this innovative technique. To our acquaintance, this is the first report of one-step RT-RPA using a basic RNA extract derived through cellular disc paper that is significantly sensitive and precise for PVS detection in potatoes. The advantages of one-step RT-RPA in terms of proficiency, robustness, and the availability of a highly pure RNA template make it an attractive choice for seed accreditation, resistance breeding, and field inspections.

Effect of potato apical leaf curl disease on glycemic index and resistant starch of potato (Solanum tuberosum L.) tubers.

Tomato leaf curl New Delhi virus-potato (ToLCNDV-potato) causes potato apical leaf curl disease which severely affects nutritional parameters such as carbohydrate, protein, and starch biosynthesis thereby altering glycemic index (GI) and resistant starch (RS) of potato. ToLCNDV-potato virus was inoculated on potato cultivars (Kufri Pukhraj [susceptible]; Kufri Bahar [resistant]) and various quality parameters of potato tuber were studied. There was a significant (P < 0.01) reduction in starch, amylose and resistant starch contents in the infected tubers. However, carbohydrate and amylopectin increased significantly (P < 0.01) which contributes to increased starch digestibility reflected with high GI and glycemic load values. Besides, ToLCNDV-potato infection leads to a significant increase in reducing sugar, sucrose, amino acid and protein in potato tubers. This is a first-ever study that highlights the impact of biotic stress on GI, RS and nutritional quality parameters of potato which is a matter of concern for consumers.

Potato dry rot disease: current status, pathogenomics and management.

Potato dry rot disease caused by Fusarium species is a major threat to global potato production. The soil and seed-borne diseases influence the crop stand by inhibiting the development of potato sprouts and cause severe rots in seed tubers, table and processing purpose potatoes in cold stores. The symptoms of the dry rot include sunken and wrinkled brown to black tissue patches on tubers having less dry matter and shriveled flesh. Fungal infection accompanied by toxin development in the rotten tubers raises more concern for consumer health. The widespread dry rot causing fungal species (Fusarium graminearum) is reported to have a hemibiotrophic lifestyle. A cascade of enzymes, toxins and small secreted proteins are involved in the pathogenesis of these hemibiotrophs. With the availability of the genome sequence of the most devastating species Fusarium sambucinum, it is important to identify the potential pathogenicity factors and small secreted proteins that will help in designing management strategies. Limited resistant cultivars and the emergence of fungicide-resistant strains have made it more threatening for potato cultivation and trade. Several novel fungicide molecules (Azoxystrobin, chlorothalonil and fludioxonil), are found very effective as tuber treatment chemicals. Besides, many beneficial bioagents and safer chemicals have shown antibiosis and mycoparasitism against this pathogen. Germplasm screening for dry rot resistance is important to assist the resistance breeding program for the development of resistant cultivars. This review aims to draw attention to the symptomatology, infection process, pathogenomics, the role of toxins and management approaches for potato dry rot disease, which is very much critical in designing better management strategies.