Molecular Characterization and Transmission Study of the Begomovirus on Hollyhock Plant Causing Leaf Curl Disease for the First Time in Province Punjab, Pakistan

I. Background: Hollyhock (Alcea rosea) is an ornamental plant belonging to the Malvaceae family and has a remarkable aesthetic and medicinal value. Previously in Pakistan, the hollyhock plant was not found to be infected by begomovirus and the plant first time showed the symptoms of typical leaf curling, puckering as well as thickened veins. II. Methods and Results: During the year 2018, symptomatic samples of the hollyhock plants were collected that exhibited characteristic typical leaf curling, puckering as well as thickened veins. DNA was extracted from the samples and the PCR technique was optimized for the detection of begomovirus followed by sequencing. The samples were detected to be infected with begomovirus by using Av/Ac core, Begomo 01/02, and CLCV 01/02 primer showed positive results with 579bp, 2.8kb, and 1.1kb nucleotide respectively. The betasatellite was amplified by using beta01/02 and CLCuMuBF11/R33 showed positive results with 1400bp and 481bp respectively. Sequencing results showed that diseased hollyhock plants were associated with Cotton leaf curl Multan virus-Rajasthan strain along with Cotton leaf curl Multan betasatellite. III. Conclusion: Hollyhock plants infected by begomovirus has been reported for the first time as a possible source of virus inoculum from Pakistan.

Tobacco curly shoot virus Down-Regulated the Expression of nbe-miR167b-3p to Facilitate Its Infection in Nicotiana benthamiana

Tobacco curly shoot virus (TbCSV) belongs to the genus Begomovirus of the family Geminiviridae, and causes leaf curling and curly shoot symptoms in tobacco and tomato crops. MicroRNAs (miRNAs) are pivotal modulators of plant development and host-virus interactions. However, the relationship between TbCSV infection and miRNAs accumulation has not been well investigated. The present study was conducted to analyze different expressions of miRNAs in Nicotiana benthamiana in response to the infection of TbCSV via small RNAs sequencing. The results showed that 15 up-regulated miRNAs and 12 down-regulated miRNAs were differentially expressed in TbCSV infected N. benthamiana, and nbe-miR167b-3p was down-regulated. To decipher the relationship between nbe-miR167b-3p expression and the accumulations of TbCSV DNA, pCVA mediation of miRNA overexpression and PVX based short tandem target mimic (STTM) were used in this study. It was found that overexpression of nbe-miR167b-3p attenuated leaf curling symptom of TbCSV and decreased viral DNA accumulation, but suppression of nbe-miR167b-3p expression enhanced the symptoms and accumulation of TbCSV. PRCP, the target gene of nbe-miR167b-3p, was silenced in plants using VIGS and this weakened the viral symptoms and DNA accumulation of TbCSV in the plants. Overall, this study clarified the effect of nbe-miR167b-3p on plant defense during TbCSV infection, and provided a framework to reveal the molecular mechanisms of miRNAs between plants and viruses.

The FBA Motif-Containing Protein NpFBA1 Causes Leaf Curling and Reduces Resistance to Black Shank Disease in Tobacco

Plant leaf morphology has a great impact on plant drought resistance, ornamental research and leaf yield. In this study, we identified a new gene in Nicotiana plumbaginifolia, NpFBA1, that causes leaf curl. The results show that the NpFBA1 protein contains only one unique F-box associated (FBA) domain and does not have an F-box conserved domain. Phylogenetic analysis placed this gene and other Nicotiana FBA genes on a separate branch, and the NpFBA1 protein localized to the nucleus and cytoplasm. The expression of NpFBA1 was induced by black shank pathogen (Phytophthora parasitica var. nicotianae) infection and treatment with salicylic acid (SA) and methyl jasmonate (MeJA). NpFBA1-overexpressing transgenic lines showed leaf curling and aging during the rosette phase. During the bolting period, the leaves were curly and rounded, and the plants were dwarfed. In addition, NpFBA1-overexpressing lines were more susceptible to disease than wild-type (WT) plants. Further studies revealed that overexpression of NpFBA1 significantly downregulated the expression of auxin response factors such as NtARF3 and the lignin synthesis genes NtPAL, NtC4H, NtCAD2, and NtCCR1 in the leaves. In conclusion, NpFBA1 may play a key role in regulating leaf development and the response to pathogen infection.

First Report of Ranunculus white mottle ophiovirus in Slovenia in Pepper with yellow leaf curling symptom and in Tomato

Pepper (Capsicum annuum) and Tomato (Solanum lycopersicum) plants showing virus-like disease symptoms were collected in 2017, 2019, and 2020, in different parts of Slovenia (Supplementary Figure 1). Total RNA was extracted from leaf tissue of individual samples using RNeasy Plant Mini kit (Qiagen) and pooled in four composite samples as follows: 2 pepper plants from 2017 (D2017), 5 pepper and 4 tomato plants from 2019 (D2019_P1), 7 tomato plants (D2020_P1), and 2 pepper and 4 tomato plants (D2020_P3) from 2020. The pooled RNA samples were sequenced using Illumina platforms, details of the sequencing experiments are in Supplementary Table 1. Reads were analyzed using CLC Genomics Workbench (v. 20.0, Qiagen) following the pipelines for plant virus discovery (Pecman et al., 2017). Reads and contigs mapping to Ranunculus white mottle ophiovirus (RWMV, GenBank accession no. AY542957 or NC_043389) were detected in all pools. The longest contig (1,255 bp) was obtained from the 2019 composite sample, mapping to the coat protein-coding RNA 3 segment of the RWMV genome (accession no. AY542957). Details of mapping, genome coverage, and other viruses detected in the pools are summarized in the Supplementary Table 1. To identify individual RWMV-infected plants from the pools, primers were designed for detection by reverse transcription-polymerase chain reaction (RT-PCR) targeting the coat protein gene (see Supplementary Table 2). Two pepper samples from two different farms, collected in 2017 and 2019 in southwest Slovenia, and four tomato samples from two different farms, collected in 2020 in central Slovenia tested positive for RWMV in RT-PCR assays. To assess the diversity of RWMV isolates, amplicons were purified using QIAquick PCR purification kit (Qiagen) and sent for Sanger sequencing. Based on maximum likelihood phylogenetic analysis, RWMV Italian and Slovenian isolates form a monophyletic clade within the genus (see Supplementary Figure 2). Pairwise nucleotide identities of the Slovenian isolates (accession no. MZ507604-MZ507609), relative to the original Italian isolate coat protein (accession no. AY542957) range from 92-97%, indicating a moderate level of diversity among isolates (see Supplementary Figure 2). Since only RWMV, bell pepper alphaendornavirus (BPEV), and pepper cryptic virus 2 (PepCV2), were present in a pepper sample from 2017, and BPEV and PepCV2 infection in pepper are not known to be associated with any of the disease symptoms (Okada et al., 2011; Saritha et al., 2016), the symptoms observed on this plant might be associated with RWMV infection. We observed mottling with interveinal chlorosis or yellowing, slight to full curling of leaves from lamina inward, as well as necrotic and aborted flowers on this plant (see Supplementary Figure 1). We cannot easily associate observed symptoms with RWMV in RWMV-positive tomatoes, since several viruses were detected in the pools containing these samples. Nevertheless, the prominent symptoms in tomato were mottling with interveinal chlorosis and leaf curling, similar to those observed in pepper. RWMV was discovered and characterized in buttercup (Ranunculus asiaticus), and detected in anemone (Anemone coronaria), from Italy (Vaira et al., 1996, 1997, 2000, 2003). It was recently detected in pepper from Australia showing veinal yellowing (Gambley et al., 2019). Here, we detected RWMV for the first time in Slovenia, and reported its first detection in tomato and pepper from Europe. These findings call for further studies on the effects of RWMV infection on tomato and pepper production, and its monitoring in neighboring European countries. Acknowledgment This study received funding from the Administration of the Republic of Slovenia for Food Safety, Veterinary Sector and Plant Protection, Slovenian Research Agency (ARRS) core financing (P4-0165), and the Horizon 2020 Marie Skłodowska-Curie Actions Innovative Training Network (H2020 MSCA-ITN) project “INEXTVIR” (GA 813542), under the management of the European Commission-Research Executive Agency. References Gambley, C., et al. 2019. New Dis. Rep. 40:13. doi:10.5197/j.2044-0588.2019.040.013. Okada, R., et al. 2011. J. Gen. Virol. 92:2664-2673. doi:10.1099/vir.0.034686-0. Pecman, A., et al. 2017. Front. Microbiol. 8:1-10. doi:10.3389/fmicb.2017.01998. Saritha, R. K., et al. 2016. VirusDisease 27:327-328. doi:10.1007/s13337-016-0327-7. Vaira, A. M., et al. 2003. Arch. Virol. 148:1037-1050. doi:10.1007/s00705-003-0016-x. Vaira, A. M., et al. 1996. Acta Hortic., 432:36-43. doi:10.17660/ActaHortic.1996.432.3. Vaira, A. M., et al. 1997. Arch. Virol. 142:2131-2146. doi:10.1007/s007050050231. Vaira, A. M., et al. 2000. Plant Dis. 84:1046-1046. doi:10.1094/PDIS.2000.84.9.1046B.

To Assess the Impact of Insecticidal Spray on Leaf Curling Caused Due to Sucking Pest and Phytotoxic Effect of Higher Doses of Insecticides in Chilli

The investigation was carried out at Samajik Vigyan Kendra, Dr. B.R. Ambedkar University, Bordi, Sehore (M.P.)-INDIA during kharif 2018-19. To assess the impact of insecticidal spray on leaf curling caused due to sucking pest and phytotoxic effect of higher doses of insecticides in chilli.The bio-efficacy of three different insecticides, namely (i) Chlorfenapyr 240 SC - spray four time with different-different doses, (ii) Fipronil 5% SC and (iii) Imidacloprid 17.8 SL. One untreated plot was also used to investigate against leaf curl and phytotoxic effect on chilli. Among these insecticides, Chlorfenapyr 240SC doses 288 g.a.i/hac (gram active ingredient per hactare) has least leaf curl indications (9.68%). It’s most effective insecticides in chilli. The least impact of leaf curl recorded in treatment T4- (9.68%) followed by T3- chlorofenapyr (11.88%),T5- Fipronil 5% SC (14.46%), T6- Imidacloprid (16.68%), T2- chlorofenapyr (17.69%) and the most elevated twisting in T7- untreated control (56.29). Further, the phytotoxic effect of treatment T1 - chlorfenapyr and T2 - chlorfenapyr were connected contrasting and T3 - untreated control. In these tried portions no phytotoxic impact likes chlorosis, Epinasty, Necrosis, Scorching , wilting and hyponasty were seen at various interim of perceptions against Chilli crop. The chilli yield was also noted highest in highest dose of T4 (16.0 tonnes ha-1) followed by second highest dose of T3 (15.4 tonnes ha-1), however, it was recorded lowest in untreated control (8.0 tonnes ha-1). The C:B benefit ratio was noted higher in T5- fipronil 5% SC@ 10 g.a.i ha-1 (3.20) followed by T6- imidacloprid 17.8 SL @ 50 g.a.i ha-1 (2.99).

Degradome Analysis of Tomato and Nicotiana benthamiana Plants Infected with Potato Spindle Tuber Viroid

Viroids are infectious non-coding RNAs that infect plants. During infection, viroid RNAs are targeted by Dicer-like proteins, generating viroid-derived small RNAs (vd-sRNAs) that can guide the sequence specific cleavage of cognate host mRNAs via an RNA silencing mechanism. To assess the involvement of these pathways in pathogenesis associated with nuclear-replicating viroids, high-throughput sequencing of sRNAs and degradome analysis were carried out on tomato and Nicotiana benthamiana plants infected by potato spindle tuber viroid (PSTVd). Both hosts develop similar stunting and leaf curling symptoms when infected by PSTVd, thus allowing comparative analyses. About one hundred tomato mRNAs potentially targeted for degradation by vd-sRNAs were initially identified. However, data from biological replicates and comparisons between mock and infected samples reduced the number of bona fide targets—i.e., those identified with high confidence in two infected biological replicates but not in the mock controls—to only eight mRNAs that encode proteins involved in development, transcription or defense. Somewhat surprisingly, results of RT-qPCR assays revealed that the accumulation of only four of these mRNAs was inhibited in the PSTVd-infected tomato. When these analyses were extended to mock inoculated and PSTVd-infected N. benthamiana plants, a completely different set of potential mRNA targets was identified. The failure to identify homologous mRNA(s) targeted by PSTVd-sRNA suggests that different pathways could be involved in the elicitation of similar symptoms in these two species. Moreover, no significant modifications in the accumulation of miRNAs and in the cleavage of their targeted mRNAs were detected in the infected tomato plants with respect to the mock controls. Taken together, these data suggest that stunting and leaf curling symptoms induced by PSTVd are elicited by a complex plant response involving multiple mechanisms, with RNA silencing being only one of the possible components.

Species composition of aphids (order Homoptera, family Aphididae) in fruit agrocenoses of the Crimea

Nine species of aphids have been identified in fruit plantations in the Crimea. The species composition and percentage varies depending on the zoning of the plantings. The correlation coefficient between the hydrothermal conditions of April-June and the number of green apple aphids r=0,55, and gray apple (rosy leaf-curling) aphids - r=0,46. Mass reproduction of apple aphids was recorded in May, which was facilitated by dry and warm weather. Low temperatures in April during the beginning of hatching of larvae restrained the growth of the number of phytophages and provoked the almost complete death of the woolly aphid population.

Effect of Water Stress on Reproduction and Colonization of Podosphaera aphanis of Strawberry

In a number of pathosystems involving the powdery mildews (Erysiphales), plant stress is associated with decreased disease susceptibility and is detrimental to pathogen growth and reproduction. However, in strawberry, anecdotal observations associate severe powdery mildew (Podosphaera aphanis) with water stress. In a 2017 survey of 42 strawberry growers in Norway and California, 40 growers agreed with a statement that water-stressed strawberry plants were more susceptible to powdery mildew compared with nonstressed plants. In repeated in vitro and in vivo experiments, we found that water stress was consistently and significantly unfavorable to conidial germination, infection, and increases in disease severity. Deleterious effects on the pathogen were observed from both preinoculation and postinoculation water stress in the host. Soil moisture content in the range from 0 to 50% was correlated (R2 = 0.897) with germinability of conidia harvested from extant colonies that developed on plants growing at different levels of water stress. These studies confirm that P. aphanis fits the norm for biotrophic powdery mildews and hosts under stress. Mild water stress, compared with a state of optimal hydration, is likely to decrease rather than increase susceptibility of strawberry to P. aphanis. We believe it is possible that foliar symptoms of leaf curling due to diffuse and inconspicuous infection of the lower leaf surfaces by P. aphanis could easily be mistakenly attributed to water stress, which we observed as having a nearly identical leaf curling symptom in strawberry.

Efficient Physiological and Nutrient Use Efficiency Responses of Maize Leaves to Drought Stress under Different Field Nitrogen Conditions

Inadequate water and nitrogen (N) supplies can limit the productivity of maize. Climate change will likely increase drought in many regions on a global scale. The determination of N fertilizer rates under field drought conditions will be critical toward the reduction of agricultural risk. For this study, drought-resistant/sensitive cultivars were selected as experimental samples. Our results revealed that drought stress reduced the relative water content (RWC) of leaves, which resulted in leaf curling, while decreasing photosynthesis levels and N accumulation. In contrast to those without N treatments, the application of N significantly increased grain yields by 26.8% during the wet year but increased only by 5.4% during the dry year. Under the same N levels, the reduction in yield caused by drought increased with the increased application of N. This was because the application of the N fertilizer translated to increase the leaf area and transpiration, exacerbated the soil water loss and induced a leaf curling state in maize, which had deleterious effects on photosynthesis and N absorption. During the dry year, the yields of drought-sensitive cultivars were even less than those without the application of N. Compared with those of drought-sensitive cultivars, the RWCs of drought-resistant cultivars decreased more rapidly, and they entered the state of leaf curling earlier. Thus, N fertilizer inputs should be reduced, and the extent of N fertilization for drought-sensitive cultivars should be reduced even further.

Suppression of nbe-miR1919c-5p Expression in Nicotiana benthamiana Enhances Tobacco Curly Shoot Virus and Its Betasatellite Co-Infection

MicroRNAs (miRNAs) are non-coding but functional RNA molecules of 21–25 nucleotides in length. MiRNAs play significant regulatory roles in diverse plant biological processes. In order to decipher the relationship between nbe-miR1919c-5p and the accumulations of tobacco curly shoot virus (TbCSV) and its betasatellite (TbCSB) DNAs, as well as viral symptom development, we investigated the function of nbe-miR1919c-5p during TbCSV and TbCSB co-infection in plants using a PVX-and a TRV-based short tandem target mimic (STTM) technology. Suppression of nbe-miR1919c-5p expression using these two technologies enhanced TbCSV and TbCSB co-infection-induced leaf curling symptoms in Nicotiana benthamiana plants. Furthermore, suppression of nbe-miR1919c-5p expression enhanced TbCSV and TbCSB DNA accumulations in the infected plants. Our results can advance our knowledge on the nbe-miR1919c-5p function during TbCSV and TbCSB co-infection.