Kaolinite nanoclay-shielded dsRNA drenching for management of Globodera pallida: An environmentally friendly pest management approach.

Globodera pallida, an obligate sedentary endoparasite, is a major economic pest that causes substantial potato yield losses. This research aimed to study the effects of gene silencing of three FMRFamide-like peptides (FLPs) genes to reduce G. pallida infestation on potato plants by using kaolinite nanoclay as a carrier to deliver dsRNAs via drenching. A dsRNA dosage of 2.0 mg/ml silenced flp-32c by 89.5%, flp-32p by 94.6%, and flp-2 by 94.3%. J2s incubated for 5 and 10 h showed no phenotypic changes. However, J2s of G. pallida efficiently uptake dsRNA of all targeted genes after 15 h of incubation. On the other hand, J2s that had been kept for 24 h had a rigid and straight appearance. Under fluorescence microscopy, all dsRNA-treated nematodes showed fluorescein isothiocyanate (FITC) signals in the mouth, nervous system, and digestive system. The untreated population of J2s did not show any FITC signals and was mobile as usual. The drenching of potato cultivar Kufri Jyoti with the dsRNA-kaolinite formulations induced deformation and premature death of J2s, compared with untreated J2s that entered J3 or J4 stages. This study validates that the nanocarrier-delivered RNAi system could be employed effectively to manage G. pallida infestations.

Development and evaluation of loop-mediated isothermal amplification assay for rapid and sensitive detection of potato cyst nematode, Globodera pallida from soil.

Potato cyst nematodes, Globodera pallida and G. rostochiensis, are economically important and difficult to manage pests of the potato crop. The cyst of both the species looks similar and it is difficult to differentiate once it turns brown upon maturity. Early detection of the PCN at the species level is crucial to avoid its further spread and for adopting the appropriate management strategies. Therefore, in the present study, highly specific and sensitive loop-mediated isothermal amplification (LAMP) assay was developed to amplify mitochondrial-Sequence Characterized Amplified Region (SCAR) sequence of potato cyst nematode, G. pallida. The LAMP assay was completed within a shorter incubation period of 60 min at 60 °C followed by the reaction termination at 80 °C for 5 min. The developed LAMP assay exhibited high specificity for G. pallida and did not detect any other species including its sibling species, G. rostochiensis. In sensitivity tests, the assay detected G. pallida at 1000 times less DNA concentration (10 fg/µl) as compared to conventional PCR (10 pg/µl). In addition to this, the developed LAMP assay was tested for the detection of G. pallida directly from the soil samples, and even a single cyst mixed with soil was successfully detected by the developed assay. Moreover, the utility of low-cost instruments like hot water bath was also demonstrated for the detection of G. pallida from the soil. The developed LAMP is a rapid, highly specific, sensitive, and cost-effective technique for the species-specific detection of G. pallida. The developed assay will facilitate the rapid detection of G. pallida at quarantine stations as well as from the fields which will help to stop its further spread in new areas and also to devise effective management strategies for sustainable potato production. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03542-x.

Chaetomium globosum KPC3: An Antagonistic Fungus Against the Potato Cyst Nematode, Globodera rostochiensis.

The potato cyst nematode (Globodera rostochiensis) is one of the most economically important pests of potato (Solanum tuberosum L.), causing significant economic losses worldwide. The identification of biocontrol agents for the sustainable management of G. rostochiensis is crucial. In this study, a potential biocontrol agent, Chaetomium globosum KPC3, was identified based on sequence analysis of the DNA internal transcribed spacer (ITS) region, the translation elongation factor 1-alpha (TEF1-α) gene, and the second largest subunit of the RNA polymerase II (RPB2) gene. The pathogenicity test of C. globosum KPC3 against cysts and second-stage juveniles (J2s) revealed that fungus mycelium fully parasitized the cyst after 72 h of incubation. The fungus was also capable of parasitizing the eggs inside the cysts. The culture filtrate of C. globosum KPC3 caused 98.75% mortality in J2s of G. rostochiensis after 72 h of incubation. The pot experiments showed that the combined application of C. globosum KPC3 as a tuber treatment at a rate of 1 lit kg-1 of tubers and a soil application at a rate of 500 ml kg-1 of farm yard manure (FYM) resulted in significantly lesser reproduction of G. rostochiensis compared to the rest of the treatments. Altogether, C. globosum KPC3 has the potential to be used as a biocontrol agent against G. rostochiensis and can be successfully implemented in integrated pest management programs.

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.

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.

A new record of Asia II 5 genetic group of Bemisia tabaci (Gennadius) in the major potato growing areas of India and its relationship with tomato leaf curl New Delhi virus infecting potato.

The whitefly, Bemisia tabaci (Gennadius), is responsible for significant yield losses in many crops, including potato, by sucking the phloem sap and transmitting a number of plant viruses. B. tabaci is a complex of cryptic species which is commonly designated as genetic groups. The B. tabaci genetic groups differ biologically with respect to host plant preference, insecticidal resistance, reproduction capacity, and ability to transmit begomoviruses. Therefore, understanding genetic variation among populations is important for establishing crop-specific distribution profile and management. We sequenced the mitochondrial cytochrome oxidase I (mtCOI) gene of B. tabaci collected from major potato growing areas of India. BLAST analysis of the 24 mtCOI sequences with reference Gene Bank sequences revealed four B. tabaci genetic groups prevailing in this region. mtCOI analysis exhibited the presence of Asia II 1, Asia II 5, Asia 1, and MEAM1 B. tabaci genetic groups. Our study highlighted that a new genetic group Asia II 5 has been detected in Indo-Gangetic Plains. Further virus-vector relationship study of ToLCNDV with Asia II 5 B. tabaci revealed that females are efficient vector of this virus as compared to males. This behavior of females might be due to their ability to acquire more virus titer than males. This study will help in better understanding of whitefly genetic group mediated virus diseases.

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.

Rapid and sensitive detection of potato virus X by one-step reverse transcription-recombinase polymerase amplification method in potato leaves and dormant tubers.

Potato virus X (PVX), is a serious threat to global potato production. A simple and rapid detection method is imperative for PVX diagnosis and early management. In this study, an isothermal one-step reverse transcription-recombinase polymerase amplification (RT-RPA) method was optimized for the quick and convenient detection of PVX in potato leaves and tubers. Our results revealed that this one-step RT-RPA method was highly efficient than the conventional reverse transcription-polymerase chain reaction (RT-PCR). The amplification reaction was free from cross-reactivity with other common potato viruses and completed within 30 min. Moreover, this RT-RPA assay did not require a thermocycler based specific temperature phase amplification and can be easily performed using a simple heating block or water bath at a temperature range of 39-42 °C. The sensitivity assay demonstrated that the developed one-step RT-RPA method was 100 times more sensitive than a routine one-step RT-PCR. Initially, the purified total RNA as the template isolated from infected leaves of potato was used for the detection of PVX. One-step RT-RPA was later performed using cellular disc paper-based simple RNA extract as a template that could detect the virus more efficiently than purified total RNA. The performance of the one-step RT-RPA assay was further evaluated using 500 field samples of leaves and tubers representing different cultivars and geographical regions. To our knowledge, this is the first report of rapid, sensitive, and reliable detection of PVX infection by one-step RT-RPA using cellular disc paper-based simple RNA extract from leaves and dormant tubers of potato. It is superior to the common RT-PCR assay in terms of its versatility, quickness, and independence of highly purified RNA template and can be adopted as a substitute to RT-PCR as an effective technique for seed potato certification, quarantine, breeding, and field surveys.

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.

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.

Development and application of fluorescent loop mediated isothermal amplification technique to detect Phytophthora infestans from potato tubers targeting ITS-1 region.

The goal of this study was to develop a fluorescent based loop-mediated isothermal amplification (LAMP) assay for a simple, sensitive and visual detection of P. infestans from tubers targeting a novel internal transcribed spacer 1 (ITS-1) region of ribosomal DNA. The ITS-1 LAMP primers were designed using the Primer Explorer V4 software. The optimization of LAMP reaction conditions and reagents concentrations were carried out with time, temperature, MgSO4, dNTPs and WarmStart Bst DNA polymerase. The amplified products were analysed using SYBR Green I dye and by agarose gel electrophoresis. We optimized reaction conditions included reagent mix, incubated at 65 °C for 60 min. The target specificity of primers was assessed with PCR, restriction digestion and sequence analysis. The developed LAMP assay was evaluated for its analytical specificity, sensitivity and validation in field tuber samples. The analytical specificity of LAMP primers indicates positive reaction with P. infestans and closely related species except P. erythosepctica. We were able to detect down to 1 pg/µl of DNA using the newly developed LAMP primers whereas the minimal amount detectable for conventional PCR was 0.1 ng/µl. Further, the samples with positive reaction developed a characteristic fluorescent green color. The detection of LAMP assay for inoculum of P. infestans was determined in the artificially inoculated leaves and tubers. In 98 field tuber samples, 54 (55.10%) were confirmed as positive by LAMP while 39 (39.79%) positive by PCR. The LAMP assay developed in this study has a potential to be a beneficial tool in early detection of P. infestans in low cost laboratory. Because the LAMP assay performed well in aspects of sensitivity, repeatability, target specificity, reliability, and visibility, it is suitable for detection of P. infestans in infected potato tubers.