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Silverleaf whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), is one of the most notorious invasive insect pests, infesting more than 900 species of plants and spreading more than 200 viral diseases. This polyphagous agricultural pest harbours diverse bacterial communities in its gut, which perform multiple functions in whiteflies, including nutrient provisioning, amino acid biosynthesis, and virus transmission. The present exploratory study compares the bacterial communities associated with silverleaf whitefly infesting cassava, also known as cassava whitefly, collected from two different zones (zone P: plains; zone H: high ranges), from Kerala, India, using next-generation sequencing of 16S rDNA. The data sets for these two regions consisted of 1 321 906 and 690 661 high-quality paired-end sequences with mean length of 150 bp. Highly diverse bacterial communities were present in the sample, containing approximately 3513 operational taxonomic units (OTUs). Sequence analysis showed a marked difference in the relative abundance of bacteria in the populations. A total of 16 bacterial phyla, 27 classes, 56 orders, 91 families, 236 genera, and 409 species were identified from the P population, against 16, 31, 60, 88, 225, and 355, respectively, in the H population. Arsenophonus sp. (Enterobacteriaceae), which is important for virus transmission by whiteflies, was relatively abundant in the P population, whereas in the H population Bacillus sp. was the most dominant group. The association of whitefly biotypes and secondary symbionts suggests a possible contribution of these bacteria to host characteristics such as virus transmission, host range, insecticide resistance, and speciation.

Elephant foot yam (Amorphophallus paeoniifolius (Dennst.) Nicolson), is an important edible tropical tuber crop, belonging to the family Araceae. Corms produced by this plant is very big and they are rich in starch, protein, mineral, vitamins, and dietary fiber but has acridity problem. This crop is susceptible to virus and phytoplasma diseases which affects crop growth and corm yield. Even though this crop has high commercial value, the problems like susceptibility to viral diseases, acridity problems, and lack of genetic diversity made hindrance in their exploitation. These issues can be resolved only by improving the characters through genetic transformation. To achieve genetic transformation in this important crop, a study was conducted to optimize various parameters for efficient Agrobacterium-mediated genetic transformation using embryogenic calli with vectors having gus reporter gene. Calli were developed using petiole and leaves of in vitro plantlets of elephant foot yam cultivar Gajendra and experiments were conducted to evaluate the sensitivity of calli to different doses of antibiotics viz. geneticin, hygromycin, ticarcillin. It was observed that complete death and discoloration of the calli were obtained with 25 mgl(−1) geneticin and 10 mgl(−1) hygromycin. The lowest lethal concentration of ticarcillin against Agrobacterium growth was found to be 500 mgl(−1) which did not affect calli growth. Optimized parameters for efficient transformation in elephant foot yam include 100 μM acetosyringone concentration with 2 days of co-cultivation at temperature 22 °C using LBA4404 strain. The putative transformants were characterized for the integration of the gus gene using PCR and nucleic acid spot hybridization. The optimized protocol is simple and reproducible and may be adapted for other cultivars also. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02824-6.

The ever-increasing demand for food production proportionate to exponential growth of global population evoked the need for applying innovative techniques for developing disease-resistant crop varieties, as the pest and pathogen attack causes considerable yield loss which in turn putts the agriculture sector and crop production in crisis. This is highly significant when the resistance conferred by conventional measures, like artificial hybridization, mutation breeding, marker-assisted selection, etc. appears to be inadequate in many cases, especially due to the evolution of new/more virulent strains/pathovars/isolates of pathogens and their unexpected host range expansion. Development of recombinant DNA technology, transgenic expression and RNA silencing strategies lead to a new era of transgenically engineered resistance in several crop species, many of which succeeded in field trials and got commercialized. Elucidation of genetic and molecular mechanisms underlying host-pathogen interactions, resistance, susceptibility and different levels of plant immune responses (PTI, ETI, HR, etc.) revealed the key genes in the host as well as pathogens that can be manipulated by transgenic approaches/techniques for conferring effective resistance. Reprogramming of phytohormonal regulatory pathways determining defence response and remodeling of molecular receptors/transcription factors involved in resistance or disease development can be brought about by transgenic methods to enhance the resistance in host plants. Targeting of conserved sequences or molecular components could provide broad-spectrum resistance in certain cases. Mining of R genes, transgenic expression of foreign R gene, etc. are other useful strategies. Potential of genome editing based on engineered nucleases like ZFNs, TALENs and CRISPR/Cas9 to precisely mutate the genomic sequence of interest can be exploited for specific targeting of the host/pathogenic genes associated with the biotic stress response. Application of all these approaches for the management of plant pathogens is discussed in this chapter.

A loop-mediated isothermal amplification (LAMP) technique was employed to develop a simple and rapid method for the detection of tomato leaf curl Bangalore virus (ToLCBaV) in diseased plants of tomato (Solanum lycopersicum). Six sets of primers were designed for LAMP technique targeting the conserved AC1 region and successfully detected ToLCBaV. No reaction was detected in the tissues of healthy plants by either the LAMP or the polymerase chain reaction (PCR). The LAMP products can be visualized by presence or absence of turbidity and staining (0.2 μL for 25 μL LAMP product) directly in the tube with nucleic acid stain dye which allowed easy detection. Sensitivity of LAMP assay is 100 times of conventional PCR technique. Although, both the LAMP and the PCR methods were capable of detecting ToLCBaV in infected tissues of tomato, the LAMP method would be more useful than the PCR method for detection of ToLCBaV infection in tomato plants because it is more rapid, simple and accurate method.

Cassava Mosaic virus Disease (CMD) is the most severe and widespread virus infection that affects cassava (Manihot esculenta Crantz) crops. This paper investigates the application of photochemical reflectance index (PRI) imaging to detect and assess the impact of varying levels of CMD infection in cassava. Towards this, narrow band reflectance images of field-grown cassava plants were recorded at 531 and 571 nm by proximal sensing with a multispectral imaging system (MSIS). It was observed that the PRI values increase with increasing levels of CMD infection in all the varieties of cassava studied. A scatter plot of the PRI image intensity yielded a sensitivity of 85 % and specificity of 79 % for discriminating visibly no CMD from initial CMD and a sensitivity of 93 % and specificity of 92 % for discriminating initial CMD from advanced CMD. Area under the receiver operator characteristics (AUC-ROC) curve was used to discriminate the CMD infection level by differentiating visibly no CMD from initial CMD [AUC = 0.92] and initial CMD from advanced CMD [AUC = 0.99]. It was observed that PRI values determined from the experimental data follow a linear inverse relationship with net photosynthetic rate (Pn) (R 2 = 0.76) and total leaf chlorophyll (Chl) content (R 2 = 0.80). The results show that PRI imaging can be utilized to discriminate healthy plants from CMD and other stress infected crops by proximal sensing in outdoor plants.

Dasheen mosaic virus (DsMV) the main causative agent of mosaic disease in elephant foot yam (Amorphophallus paeoniifolius) belongs to Potyviridae. The complete genome sequence of DsMV infecting A. paeoniifolius in Kerala state, India was assembled from the whole transcriptome sequencing reads of diseased host samples. The sequence of the virus which showed 83% identity with DsMV infecting Zantedeschia aethiopica (China) was confirmed through amplification and sequencing with primers designed based on the assembled sequence. Unambiguous recombination events mainly confined to the 5′ terminal 4000 nucleotides of the genome with phylogenetically related potyviruses were recognized. The length and genome composition towards the 3′ end comprising NIb, CP and 3′ UTR were not dependent on the host or the source region.

The most characterised Dasheen mosaic virus and many other unreported putative viruses are involved in the mixed viral mosaic infection of elephant foot yam (Amorphophallus paeoniifolius). The in vitro propagation of corm bud tips for virus free plantlet production was carried out in three different culture phases consisting of callusing, shoot regeneration and rooting. A 100 % survival rate was recorded on hardening in sand: soil: coir pith (1:1:1) mixture. A total of 84 % of regenerated plantlets were found to be virus free on indexing of 21 in vitro lines with species specific/genus specific serological and molecular diagnostic techniques. Transcriptome sequencing was carried out for two randomly selected in vitro plants and a mosaic infected field sample. Not any of the known potyviruses were traced in the transcriptome profiles of supposed virus free plants thus confirming the complete potyviruses elimination. Disease symptoms or re-occurrence was not observed in the hardened virus-free lines of the plant.

UMR BFP - Equipe Virologie; International audience; We here assessed the capability of the MinION sequencing approach to detect and characterize viruses infecting a water yam plant. This sequencing platform consistently revealed the presence of several plant virus species, including Dioscorea bacilliform virus, Yam mild mosaic virus and Yam chlorotic necrosis virus. A potentially novel ampelovirus was also detected by a complimentary Illumina sequencing approach. The full-length genome sequence of yam chlorotic necrosis virus was determined using Sanger sequencing, which enabled determination of the coverage and sequencing accuracy of the MinION technology. Whereas the total mean sequencing error rate of yam chlorotic necrosis virus-related MinION reads was 11.25%, we show that the consensus sequence obtained either by de novo assembly or after mapping the MinION reads on the virus genomic sequence was >99.8% identical with the Sanger-derived reference sequence. From the perspective of potential plant disease diagnostic applications of MinION sequencing, these degrees of sequencing accuracy demonstrate that the MinION approach can be used to both reliably detect and accurately sequence nearly full-length positive-sense single-strand polyadenylated RNA plant virus genomes.

The phytohormone auxin is involved in the cell division, proliferation and initial thickening of storage root of sweet potato. This article reports the differential expression of functionally distinct auxin responsive candidate genes such as Auxin Response Factors (ARF) and Auxin/Indole 3-Acetic Acid (Aux/IAA) in the storage root of sweet potato [Ipomoea batatas (L.) Lam]. The differential expression of ESTs of these auxin regulated genes were analyzed in the storage root of sweet potato as compared to non-storage root using the Gene Expression Hybridization kit (Part Number 5190-0404; Agilent). During the initial storage root development of sweet potato ARF1, ARF2, ARF10, ARF9 and ARF16 are proposed to be involved in regulating genes controlling cell division pattern while ARF7, ARF8 promote cell elongation/expansion and links brassinosteroid, ethylene and auxin and JA interaction, whereas ARF4 is involved in asymmetric pattern establishment. Several Aux/IAA genes, viz. OsIAA2, OsIAA7, OsIAA10, OsIAA21, OsIAA30 were up-regulated whereas, OsIAA4, OsIAA10, OsIAA17, OsIAA21, OsIAA30, OsIAA31 were down-regulated in the storage root as compared to fibrous root of sweet potato. The down-regulation of IAA4 may be significant in determining the storage root length of sweet potato.

Elephant foot yam (Amorphophallus paeoniifolius) is a vegetatively propagated stem tuber crop. In this investigation we describe a highly competent and reproducible in vitro propagation of the plant from corm bud, petiole and young leaf explants. Friable callus was initiated from all the explants on modified MS medium (half the concentration of NH4NO3 and KNO3) supplemented with 0.5 mg l-1 each of benzyl amino purine (BAP), α-naphthalene acetic acid (NAA) and 2,4-dichloro phenoxy acetic acid (2,4-D). Shoot regeneration from calli was optimal on modified MS medium supplemented with 5.0 mg l-1 BAP and 1.0 mg l-1 NAA. Microcorms, capable of producing micro shoots all over the surface, were induced from the callus at a frequency of 90% on shoot regeneration medium supplemented with 5% sucrose. Rooting was 100% on modified liquid MS medium augmented with 5.0 mg l-1 Indole 3- butyric acid (IBA). A 100% survival rate of plantlets on transplantation to soil: sand: coir pith mixture was recorded.Keywords: Callus, elephant foot yam, in vitro, microcorms, regeneration, somatic embryoAfrican Journal of Biotechnology, Vol 13(49) 4508-4514

The genus Macluravirus of the family Potyviridae currently contains six recognized and two tentative virus species. In India, so far only two macluraviruses eg., large cardamom chirke virus (LCCV) and cardamom mosaic virus (CdMV) infecting large cardamom (Amomum subulatum) and small cardamom (Elettaria cardamomum), respectively have been studied well. Recently, a new macluravirus, yam mottling virus has been tentatively identified in mild mosaic disease of yam (Dioscorea spp) in southern India. LCCV is distributed in large cardamom cultivated in the North-East sub-Himalayan mountains and CdMV in small cardamom cultivated in southern India. Both these macluraviruses cause chlorotic streak mosaic disease in cardamom. CdMV and LCCV are known in India since long time and considerable infromation has been generated. This chapter summarizes the work on the biological and molecular properties of macluraviruses occurring in India.

Carlaviruses infects field, vegetable and ornamental crops in India. The genus Carlavirus (family Betaflexiviridae) has as many as 43 recognised virus species and 13 tentative members. Only five carlavirus species, Cowpea mild mottle virus, Chrysanthemum virus B, Lily symptomless virus, Potato virus S and Garlic common latent virus and one tentative member, football lily mosaic virus are known in India. In this chapter, characterisation of carlavirus occurring in India is presented.

Occurrence of various types of viral symptoms viz. mosaic, mottling, puckering, stunting and filiformy/shoestring was noticed on Elephant foot yam in India. Immunosorbent electron microscopy of infected leaf samples with Potyvirus group–specific antiserum revealed the association of flexuous Potyvirus particles. Infected leaf samples were collected from different locations in Kerala, Orissa and Andhra Pradesh; and total RNA was extracted from infected leaves. One-step reverse transcription polymerase chain reaction assay was carried out using Potyvirus group–specific primers and an amplified product of 327 and 719 bp were obtained. Sequence analysis of nucleic acid and deduced amino acid sequences with the already available Potyvirus sequences in Genbank showed 91% and 97% similarity with Dasheen mosaic virus (DsMV) isolate DeSLK2 (accession Number AJ305434) at nucleic acid and amino acid level respectively. Comparison of 3′UTR region using universal primers MJ1 and M4T also confirmed that the virus under...

Reverse transcription polymerase chain reaction of the infected leaf samples of Colocasia esculenta plants showing severe whitish feathery symptoms were carried out using Potyvirus group specific primers, resulting in an amplicon of 327 bp, encoding the core region of the coat protein gene. Sequencing and BLAST analysis showed that the virus is distinct, closely related to Dasheen mosaic virus (DsMV). Sequence analysis revealed 86 and 96% identity at the nucleotide and amino acid level respectively with the DsMV isolate SY1(accession Number AJ628756). This is the first molecular level characterisation of the DsMV infecting C. esculenta in India.

The genetic variability in starchy Curcuma species was assessed using Random Amplified Polymorphic DNA (RAPD) technique. The RAPD pattern generated by 20 primers revealed a high degree of polymorphism. A total of 274 bands were generated of which 264 were polymorphic. All the species were separated into 3 clusters using UPGMA. C. aromatica, C. leucorrhiza, and C. brog formed a cluster within which C. longa and C. zedoaria formed a subgroup. C. harita was genetically distinct from all the other Curcuma species. Since it is difficult to distinguish different species by leaf morphology, the RAPD pattern has high utility in identification of starchy Curcuma species.

Cassava (Manihot esculenta Crantz.) is the most important vegetatively propagated food staple in Africa and a prominent industrial crop in Latin America and Asia. Its vegetative propagation through stem cuttings has many advantages, but deleteriously it means that pathogens are passed from one generation to the next and can easily accumulate, threatening cassava production. Cassava-growing continents are characterized by specific suites of viruses that affect cassava and pose particular threats. Of major concern, causing large and increasing economic impact in Africa and Asia are the cassava mosaic geminiviruses that cause cassava mosaic disease in Africa and Asia and cassava brown streak viruses causing cassava brown streak disease in Africa. Latin America, the center of origin and domestication of the crop, hosts a diverse set of virus species, of which the most economically important give rise to cassava frog skin disease syndrome. Here, we review current knowledge on the biology, epidemiology, and control of the most economically important groups of viruses in relation to both farming and cultural practices. Components of virus control strategies examined include: diagnostics and surveillance, prevention and control of infection using phytosanitation, and control of disease through the breeding and promotion of varieties that inhibit virus replication and/or movement. We highlight areas that need further research attention and conclude by examining the likely future global outlook for virus disease management in cassava.

Dasheen mosaic virus (DsMV), the pathogen causing mosaic disease of elephant foot yam (Amorphophallus paeoniifoilius) is disseminated mainly through vegetative propagation of the tubers. For the rapid and sensitive detection of the virus, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay based on the coat protein gene has been developed. A final concentration of 5.4 mM magnesium sulphate and 0.7 M betaine in the reaction mixture was found to be optimum for getting characteristic ladder like bands of the amplified product after gel electrophoresis. The reaction was set at 65°C for 50 min followed by reaction termination at 86°C for 5 min in a water bath. The sensitivity of the assay was found to be 100 times higher than that of RT-PCR. The virus was indexed successfully from tubers of elephant foot yam. In tube detection of the DsMV was carried out using fluorescence detection reagents. The assay was validated with field samples from various regions of Kerala state, India.

A reverse transcription polymerase chain reaction assay using potyvirus specific primers designed from the core of the coat protein was carried out, and a cDNA fragment of 327 bp was obtained from most of the potyviruses infecting the tropical tuber crops. Reverse transcription polymerase chain reaction (RT-PCR) products were sequenced and found to be derived from the expected virus. Specific cDNA probe was generated from the amplicon, and the probe was then successfully used for the diagnosis of the potyviruses infecting the major tuber crops through biotinylated nucleic acid spot hybridisation. The specific probe developed could detect the potyviruses infecting tuber crops namely SPFMV, DsMV and DAV from sweet potato, aroids and yams respectively.Key words: Coat protein, potyvirus, reverse transcription polymerase chain reaction, nucleic acid spot hybridisation.

The development of Catenaria anguillulae on the immobilized juveniles of Heterodera cajani was studied. Within 2 h of inoculation, the zoospores of C. anguillulae congregated at the oral openings of juveniles and in 13–15 h the germ-tube passed from the side of the stylet, growing up to the tail region. After 1 h the mycelium became septate at regular intervals and swelling of the sporangia commenced, attaining full size in 40–50 min as it touched the nematode body. At this stage, second septation occurred with rhizoids developing along with growing mycelium. After nearly 5 h of the second stage of septa formation, grouping of fat globules into groups of 4–6 occurred, along with the initiation of discharge-tubes. Precisely within 30 min, 8–12 smaller globules were rearranged in circular forms, followed by half-moon shape 17 min later. Zoospore differentiation, vesicle formation and release of zoospores occurred within 50 min of the half-moon stage.

Greater yam ( Dioscorea alata ) is an important edible species of Asia, and is mainly used as a subsidiary vegetable. Kerala, West Bengal, Bihar, Orissa, Assam, Gujarat and Maharashtra are the major yam-cultivating states in India. In 2002 leaves of D. alata growing in farmers’ fields in the Pathanamthitta district of Kerala showed black, circular, concentric spots 5–10 mm in diameter in the middle and bottom portion of the vines. As the disease advanced, the central portion of the leaf spots dried and fell out, resulting in shot-hole symptoms. When diseased leaves were placed in a humid chamber, abundant fluffy white mycelia emerged from the leaf spots. When isolated on potato dextrose agar, the diameter of hyphae ranged from 1·5 to 2·7 μ m. Numerous round to ellipsoidal, dark brown-to-black, smooth sclerotia were observed in the culture. Disease symptoms were reproduced when detached D. alata leaves were inoculated with the isolated culture and incubated in high humidity in Petri dishes for 4 days. The pathogen was reisolated from the inoculated leaves. Based on morphological and cultural characteristics (Singh, 1982), the pathogen was identified as Sclerotium rolfsii . A culture has been deposited in the Indian Type Culture Collection of the Indian Agricultural Research Institute, New Delhi (ITCC 5789·04). Sclerotium rolfsii associated with leaf spot of Dioscorea spp. has been reported earlier in Nigeria (Amusa, 1999). There are no previous records of this disease on D. alata from India (Bilgrami & Jamaluddin Rizwi, 1991; Butler, 1997).