Studies on a Nigerian isolate of banana streak badnavirus: II. Effect of intraplant variation on virus accumulation and reliability of diagnosis by ELISA

Monitoring of banana streak badnavirus (BSV) antigens and symptoms in naturally BSV-infected plantain and banana (Musa spp.) plants showed a great variation in symptom expression, distribution and relative concentration of BSV between and within plants. Expression and distribution of symptoms was erratic within individual leaves as well as between different leaves of the same plant. The concentration of BSV antigens detected by triple antibody sandwich enzyme linked immunosorbent assay (TAS-ELISA) varied in different plant parts including leaf lamina, midrib and pseudostem, roots and young ‘cigar’ leaf. The concentration of BSV antigens was high in symptomatic tissues but was low or below the limits of detection in most asymptomatic tissues. During ‘hot dry’ seasons when symptoms were not fully expressed, the concentration of BSV antigens in leaf tissues declined drastically, often below the detection limit of TAS-ELISA. These results suggested that for more reliable detection of BSV antigens by TAS-ELISA, it is advisable to index plants using composite tissue samples comprising as many leaves as possible for each plant and collected during cool and/or rainy seasons when symptom expression is generally severe.     

Studies on a Nigerian isolate of banana streak badnavirus: I. Purification and enzyme linked immunosorbent assay

A Nigerian isolate of banana streak badnavirus (BSV) was purified and a polyclonal antiserum was produced in mice. The antiserum titre was between 1:10 000 and 1:40 000 in enzyme linked immunosorbent assay (ELISA), and showed a good specificity to BSV antigens. Comparative tests were carried out to determine the sensitivity and reliability of BSV antigen detection by double antibody sandwich (DAS)-ELISA, triple antibody sandwich (TAS)-ELISA, antigen coated plate (ACP)-ELISA, and protein-A coated antibody sandwich (PAS)-ELISA. TAS-ELISA using rabbit polyclonal antiserum to trap BSV and mouse polyclonal antiserum to detect the virus particles, was more sensitive than ACP-ELISA and PAS-ELISA and detected BSV in plant extracts from both symptomatic and some asymptomatic plants. However, immunosorbent electron microscopy detected more BSV-infected plants from asymptomatic plant samples than did TAS-ELISA. Results of this study showed that detection of BSV antigens in sap extracts by TAS-ELISA was most efficient with symptomatic tissues which occurred most frequently in the ‘cool rainy’ season. This suggests that for more reliable BSV-indexing of field samples, tissue sampling should be done during the rainy season when most BSV-infected plants express severe symptoms.     

Evaluation of micropropagated plantain and banana (Musa spp.) for banana streak badnavirus incidence under field and screenhouse conditions in Nigeria

Between 1991 to 1996, more than 50 Musa hybrids and 10 landraces were evaluated under field and screenhouse conditions for virus symptoms resembling those caused by banana streak badnavirus (BSV). The symptoms included chlorotic streaks, leaf deformation, stunting, cigar leaf death, distortion of the peduncle, bunch or fruits, and internal pseudostem necrosis. Immunosorbent electron microscopy (ISEM) of randomly selected plants with one or more of these symptoms confirmed the presence of BSV particles in 15 tropical Musa plantain hybrids (TMPx) and five Musa landraces. Under both field and screenhouse conditions, the incidence of symptomatic plants in the hybrids was significantly higher than in the landraces. The hybrids also generally had a higher concentration of BSV antigens, as determined by enzyme-linked immunosorbent assay (ELISA). By contrast, most BSV-infected landraces were symptomless and had very low or undetectable amounts of BSV antigens. There was a significant variation in incidence of symptomatic plants between genotypes, experiments and year of observation. These results are discussed in relation to the higher natural BSV incidence observed on some Musa hybrids as compared with their parental genotypes.     

Molecular characterization of a new badnavirus associated with streak symptoms on enset (Ensete ventricosum,Musaceae)

Electron microscopy of leaf samples displaying streak symptoms from enset (Ensete ventricosum), a banana‐like plant widely cultivated in Ethiopia, showed the presence of bacilliform shaped virions as known for badnaviruses. DNA extracts subjected to rolling circle amplification (RCA), polymerase chain reaction (PCR) and cloning and sequence analysis revealed that the virus has a circular double‐stranded DNA genome of 7,163 nucleotides encoding predicted proteins of 21.5 kDa, 14.5 kDa and 202.5 kDa, a genome organization known for badnaviruses. The virus is phylogenetically most closely related to Sugarcane bacilliform Guadeloupe D virus with a nucleotide sequence identity of 77.2% at the conserved RT/RNase‐H region and 73.6% for the whole genome. Following the current species demarcation criteria, the virus should be considered as an isolate of a new species in the genus Badnavirus for which the name Enset leaf streak virus (ELSV) is suggested. Leaf samples from enset and banana were screened using virus‐specific primers, and ELSV was detected in six of 40 enset but not found in any of 61 banana samples. On the other hand, Banana streak OL virus (BSOLV) was detected from the majority (60%) of symptomatic banana samples but not from enset samples. This paper reports the first full‐genome sequence of a putative new badnavirus species infecting plants in the genus Ensete. In addition, this is the first report of the occurrence of BSOLV in Ethiopia.     

Molecular study of two distinct phytoplasma species associated with streak yellows of date palm in Iran

A disease with symptoms similar to palm lethal yellowing was noticed in the early 2013 in Khuzestan Province (Iran) in date palm (Phoenix dactylifera). Infected trees displaying symptoms of streak yellows and varied in the incidence and severity of yellowing. A study was initiated to determine whether phytoplasma was the causal agent. Polymerase chain reaction–restriction fragment length polymorphism (PCR‐RFLP) methods using universal phytoplasma primers pairs R16mF1/mR1 and M1/M2 were employed to detect putative phytoplasma(s) associated with date palm trees. Nested PCR using universal primers revealed that 40 out of 53 trees were positive for phytoplasma while asymptomatic date palms from another location (controls) tested negative. RFLP analyses and DNA sequencing of 16S rDNA indicated that the presence of two different phytoplasmas most closely related to clover proliferation (CP) phytoplasma (group 16SrVI) and ash yellows (AY) phytoplasma (group 16SrVII). Sequence analysis confirmed that palm streak yellows phytoplasmas in each group were uniform and to be phylogenetically closest to “CandidatusP. fraxini” (MF374755) and “Ca. P. trifolii” isolate Rus‐CP361Fc1 (KX773529). Result of RFLP analysis of secA gene of positive samples using TruI and TaqI endonuclease is in agreement with rDNA analysis. On this basis, both strains were classified as members of subgroups 16SrVI‐A and 16SrVII‐A. This is the first report of a phytoplasma related to CP and AY phytoplasma causing date palm yellows disease symptoms.     

Distribution and variability of deformed wing virus of honeybees (Apis mellifera) in the Middle East and North Africa

Three hundred and eleven honeybee samples from 12 countries in the Middle East and North Africa (MENA) (Jordan, Lebanon, Syria, Iraq, Egypt, Libya, Tunisia, Algeria, Morocco, Yemen, Palestine, and Sudan) were analyzed for the presence of deformed wing virus (DWV). The prevalence of DWV throughout the MENA region was pervasive, but variable. The highest prevalence was found in Lebanon and Syria, with prevalence dropping in Palestine, Jordan, and Egypt before increasing slightly moving westwards to Algeria and Morocco Phylogenetic analysis of a 194 nucleotide section of the DWV Lp gene did not identify any significant phylogenetic resolution among the samples, although the sequences did show consistent regional clustering, including an interesting geographic gradient from Morocco through North Africa to Jordan and Syria. The sequences revealed several clear variability hotspots in the deduced amino acid sequence, which furthermore showed some patterns of regional identity. Furthermore, the sequence variants from the Middle East and North Africa appear more numerous and diverse than those from Europe.     

Vector competence of Culicoides species and the seroprevalence of homologous neutralizing antibody in horses for six serotypes of equine encephalosis virus (EEV) in South Africa

Abstract. Field‐collected Culicoides species (Diptera: Ceratopogonidae) were fed on horse blood–virus mixtures containing one of the six serotypes of equine encephalosis virus (EEV1 to EEV6). The virus mean titres in the bloodmeals varied between 6.1 and 7.0 log₁₀TCID₅₀/mL. Of 19 Culicoides species assayed after 10 days extrinsic incubation at 23.5°C, five yielded the challenge virus, namely Culicoides (Avaritia) imicola Kieffer (EEV1–6), C. (A.) bolitinos Meiswinkel (EEV1, 2, 4, 6), C. (Meijerehelea) leucostictus Kiefer (EEV1, 2), C. (Culicoides) magnus Colaço (EEV1) and C. (Hoffmania) zuluensis de Meillon (EEV2). Virus recovery rates ranged from 0.5 to 13%. The mean levels of viral replication differed between serotypes and Culicoides species and ranged from 1.0 to 2.3 log₁₀TCID₅₀/midge. Culicoides midges shown in this study to be susceptible to oral infection with EEV are widely distributed in South Africa but differ considerably in their abundance, host preference and breeding sites. Of 1456 horses tested, 1144 (77%) had antibody to EEV. Homologous virus‐neutralizing antibodies to all six serotypes were detected in individual horses from all eight geographical provinces of South Africa. The distribution, prevalence, and the rate of exposure to individual serotypes varied significantly between regions. The potential for vectoring of EEV in the field by several Culicoides species with unique ecologies and lack of cross‐protection to re‐infection with multiple serotypes highlights some of the mechanisms that are likely to play a role in the virus' natural maintenance cycle and the highly efficient level of countrywide transmission amongst South African horses.     

Molecular diagnosis of begomovirus associated with Chilli leaf curl disease in Jeddah,Saudi Arabia

Chilli (Capsicum annum L.) is well known as ‘wonder spice’. This is a very valuable cash crop grown as a vegetable globally. Chilli leaf curl disease is a major threat and global concern for the cultivation of Chilli by farmers and growers. In this work, the molecular diagnosis, genetic diversity, phylogenetic relationship, and begomovirus association with Chilli leaf curl disease have been discussed. The infected leaves were randomly harvested from the Chilli field, at Jeddah, Saudi Arabia. A group of begomovirus vector, whiteflies were also observed on the Chilli crop and infected weeds growing in the neighboring field. The begomovirus was confirmed by coat protein gene specific primer, dot blot hybridization, sequencing and sequence analysis. The full coat protein gene was found to have 774 nucleotides. The nucleotide sequences analysis shared the highest identity with Tomato yellow leaf curl virus reported earlier infecting tomato from Saudi Arabia, and the lowest identity was observed with Tomato yellow leaf curl virus Oman isolate. The overall sequence identity ranged from more than ninety percent among the analyzed sequences. The phylogenetic relationship analysis formed the major three clusters and showed the closed clustering with Tomato yellow leaf curl virus isolates. The natural spread of the Tomato yellow leaf curl virus on the Chilli crop from other crops poses an important and serious threat to Chili cultivation in the Kingdom of Saudi Arabia. Based on the literature review and current evidence, this is the first report of leaf curl disease of Chilli from Saudi Arabia.     

Diversity of symbiotic bacteria associated with Bemisia tabaci (Homoptera: Aleyrodidae) in cassava mosaic disease pandemic areas of Tanzania

All Bemisia tabaci individuals harbour an obligate bacterial symbiont (Portiera aleyrodidarum), and many also harbour non‐essential facultative symbionts. The association of symbiotic bacteria with the various genetic groups of B. tabaci remains unknown for East Africa. This study aimed to assess any association between the various whitefly genetic groups and the endosymbionts they harbour; to investigate if a unique endosymbiont is associated with super‐abundant whiteflies, and to provide baseline information on endosymbionts of whiteflies for a part of East Africa. Whiteflies collected during surveys in Tanzania were genotyped and screened for the presence of the obligate and six secondary symbionts (SS): Rickettsia (R), Hamiltonella (H), Arsenophonus (A), Wolbachia (W), Cardinium (C) and Fritschea (F). The results revealed the presence of Mediterranean (MED), East Africa 1 (EA1), Indian Ocean (IO) and Sub‐Saharan Africa 1 (SSA1) genetic groups of Bemisia tabaci, with SSA1 further clustered into four sub‐groups: SSA1‐SG1, SSA1‐SG2, SSA1‐SG1/2 and SSA1‐SG3. F was completely absent from all of the whiteflies tested while R was always found in double or multiple infections. In general, no particular symbiont appeared to be associated with the super‐abundant SSA1‐SG1 B. tabaci, although A or AC infections were common among infected individuals. The most striking feature of these super‐abundant whiteflies, dominating cassava mosaic disease pandemic areas, was the high prevalence of individuals uninfected by any of the six SS tested. This study of the endosymbionts of B. tabaci in East Africa showed contrasting patterns of infection in crop and weed hosts.     

Mating compatibility, life-history traits, and RAPD-PCR variation in Bemisia tabaci associated with the cassava mosaic disease pandemic in East Africa

The pandemic of a severe form of cassava mosaic virus disease (CMVD) in East Africa is associated with abnormally high numbers of its whitefly vector, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae). To determine whether a novel B. tabaci biotype was associated with the CMVD pandemic, reproductive compatibility, fecundity, nymphal development, and random amplified polymorphic DNA (RAPD) variability were examined in, and between, B. tabaci colonies collected from within the CMVD pandemic and non-pandemic zone in Uganda. In a series of reciprocal crosses carried out over two generations among the six CMVD pandemic and four non-pandemic zone cassava B. tabaci colonies, there was no evidence of mating incompatibility. All the crosses produced both female and male progeny in the F₁ and F₂ generations, which in a haplo-diploid species such as B. tabaci indicates successful mating. There also were no significant differences between the sex ratios for the pooled data of experimental crosses, between individuals from two different colonies and control crosses between individuals from the same colony. Only one instance of mating incompatibility occurred in a control cross between cassava B. tabaci from Uganda and cottonB. tabaci from India. Measures of fecundity of the pandemic and non-pandemic zone B. tabaci on four cassava varieties showed no significant differences in their fecundity, nymphal development or numbers surviving to adult eclosion. Cluster analysis of 26 RAPD bands using six 10-mer primers was concordant with the mating results, grouping the pandemic and non-pandemic zone colonies into a single large group, also including a B. tabaci colony collected from cassava in Tanzania. These results suggest that it is unlikely that the severe CMVD pandemic in East Africa is associated with a novel and reproductively isolated B. tabaci biotype.     

Spider assemblages associated with leaf litter of three tree species in central South Africa (Arachnida: Araneae)

Spiders were collected by sifting leaf litter of three common tree species, Cussonia paniculata sinuata, Olea europaea africana and Searsia lancea, in an undisturbed grassland habitat in central South Africa. The study aimed to collect baseline data on spider assemblage structure, seasonality and the effects of litter structure on spider assemblages. In total, 1385 spiders were collected, representing 26 families and 56 species. Four species, Obatala sp. (Amaurobiidae, 22.7%), Camillina maun Platnick & Murphy (Gnaphosidae, 17.0%), Proevippa sp. (Lycosidae, 11.4%) and Zelotes frenchi Tucker (Gnaphosidae, 9.2%) dominated the fauna. Spider abundance was the highest in Olea litter (n = 607), followed by Searsia (n = 453) and Cussonia (n = 325). Searsia had the greatest number of species (41 spp.), followed by Olea (35 spp.) and Cussonia (30 spp.). Both spider abundance and species richness per sample varied significantly between the tree species. Of the ten most abundant species, all but two showed a significant preference for particular litter types. Mean spider abundance and species richness per sample were the lowest in Cussonia litter, which was the deepest litter type, but with the most interstitial space, whereas mean abundance and species richness per sample were the highest in Olea litter, which was the shallowest and moderately compact in structure.     

Molecular evidence for association of Cotton leaf curl Alabad virus with yellow vein mosaic disease of okra in North India

Two virus isolates (OY77 and OY81B) from okra plants showing yellow vein mosaic, downward curling and vein twisting symptoms were collected from different farmer's fields in Karnal, Haryana state, India. The genomes of the two isolates were amplified, cloned, sequenced and analysed. The analysis indicated that the isolates are similar with 89.2% nucleotide sequence identity. Based on the current threshold cut-off value for taxonomy distinguishing the genus begomoviruses species from strains, the two isolates are designated as strains of Cotton leaf curl Alabad virus (CLCuAV) which shared nucleotide sequence identity of >90% with CLCuAV infecting cotton in Pakistan. Phylogenetic and recombination analyses of the major genome component of OY77 and OY81B is derived from different begomviruses (CLCuAV, BYVMV, CLCuMuV) as the foremost parents for evolution of these new recombinant strains.     

Unusual filamentous virus-like particles in symptomless blackberry associated with severe leaf curling in the virus indicator Rubus macraei, and anomalous data using a degenerate badnavirus primer in PCR of Rubus species

Electron microscopy of ultrathin sections of leaves of symptomless Himalaya Giant blackberry and of the virus indicator species, Rubus macraei, showing severe leaf curl symptoms following graft inoculation with scions from this blackberry, detected highly flexuous virus‐like particles with an unusual ‘beaded’ structure. Such particles were restricted to a few vascular cells and were distinct from P‐protein common in some such cells. This virus, provisionally named Hawaiian rubus leaf curl virus (HRLCV), symptomlessly infected a wide range of Rubus species and cultivars. Badnavirus‐like bacilliform particles were observed in some cells of a single R. macraei plant showing leaf curl symptoms following graft inoculation with the causal agent of this disease symptom from Himalaya Giant blackberry after passage through red raspberry, but not in any other material. PCR with primer sets for the badnaviruses Rubus yellow net virus and Gooseberry veinbanding associated virus, showed that no Rubus sources studied contained these viruses. However, using a sequence‐specific primer set designed from the sequence of the product generated with a badnavirus degenerate primer set, a specific product was amplified from healthy plants of all of 16 raspberry cultivars and two Rubus species, but not from 16 blackberry cultivars (including cv. Himalaya Giant). All of these sources were free from viruses known to occur in Rubus. Sequence analysis of this product showed no homology with any known badnavirus, or with any other published sequences. It seems most likely therefore that a region of the raspberry genome has been amplified using the degenerate badnavirus primer set and that it is absent from the blackberry genome.     

Photosynthesis, water use efficiency and stable carbon isotope composition are associated with anatomical properties of leaf and xylem in six poplar species

Although fast‐growing Populus species consume a large amount of water for biomass production, there are considerable variations in water use efficiency (WUE) across different poplar species. To compare differences in growth, WUE and anatomical properties of leaf and xylem and to examine the relationship between photosynthesis/WUE and anatomical properties of leaf and xylem, cuttings of six poplar species were grown in a botanical garden. The growth performance, photosynthesis, intrinsic WUE (WUE_i), stable carbon isotope composition (δ¹³C) and anatomical properties of leaf and xylem were analysed in these poplar plants. Significant differences were found in growth, photosynthesis, WUE_i and anatomical properties among the examined species. Populus cathayana was the clone with the fastest growth and the lowest WUE_i/δ¹³C, whereas P. × euramericana had a considerable growth increment and the highest WUE_i/δ¹³C. Among the analysed poplar species, the highest total stomatal density in P. cathayana was correlated with its highest stomatal conductance (g_s) and lowest WUE_i/δ¹³C. Moreover, significant correlations were observed between WUE_i and abaxial stomatal density and stem vessel lumen area. These data suggest that photosynthesis, WUE_i and δ¹³C are associated with leaf and xylem anatomy and there are tradeoffs between growth and WUE_i. It is anticipated that some poplar species, e.g. P. × euramericana, are better candidates for water‐limited regions and others, e.g. P. cathayana, may be better for water‐abundant areas.     

Climate oscillation during the Quaternary associated with landscape heterogeneity promoted allopatric lineage divergence of a temperate tree Kalopanax septemlobus (Araliaceae) in East Asia

We investigated the biogeographic history of Kalopanax septemlobus, one of the most widespread temperate tree species in East Asia, using a combined phylogeographic and palaeodistribution modelling approach. Range-wide genetic differentiation at nuclear microsatellites (G'(ST) = 0.709; 2205 samples genotyped at five loci) and chloroplast DNA (G(ST) = 0.697; 576 samples sequenced for 2055 bp at three fragments) was high. A major phylogeographic break in Central China corresponded with those of other temperate species and the spatial delineation of the two temperate forest subkingdoms of East Asia, consistent with the forests having been isolated within both East and West China for multiple glacial-interglacial cycles. Evidence for multiple glacial refugia was found in most of its current range in China, South Japan and the southernmost part of the Korean Peninsula. In contrast, lineage admixture and absence of private alleles and haplotypes in Hokkaido and the northern Korean Peninsula support a postglacial origin of northernmost populations. Although palaeodistribution modelling predicted suitable climate across a land-bridge extending from South Japan to East China during the Last Glacial Maximum, the genetic differentiation of regional populations indicated a limited role of the exposed sea floor as a dispersal corridor at that time. Overall, this study provides evidence that differential impacts of Quaternary climate oscillation associated with landscape heterogeneity have shaped the genetic structure of a wide-ranging temperate tree in East Asia.     

Incidence of fungal species associated with leaf rot disease of coconut palms in relation to weather and the stage of lesion development

Leaf rot disease (LRD) of coconut occurs in Kerala State, India, and is generally severe during the monsoon, a time of high rainfall and r.h. and low maximum temperature. The pathogenic fungi Colletotrichum gloeosporioides, Exserohilum rostratum, Gliocladium vermoeseni, Fusarium solani, F. moniliforme var. intermedium, Thielaviopsis paradoxa and Rhizoctonia solani were among 14 species isolated from spindles of palms with LRD in multiple samplings of two experiments, each lasting 1 year. Co-occurrence of fungi was observed in 70–76% of spindles although leaf pieces yielded mostly individual fungi irrespective of the stage of lesion development. C. gloeosporioides occurred most frequently on early lesions. The incidence of C. gloeosporioides was most strongly correlated with rainfall and r.h. and, negatively, with maximum temperature and hours of sunshine. It was the fungus isolated most commonly during the monsoon and occurred on early lesions more frequently than on advanced lesions (determined in one experiment). C. gloeosporioides is thus implicated as the principal pathogen involved in initiation of the disease during monsoons. Its relatively low incidence in the dry season suggests a quiescent phase between periods of parasitic activity. E. rostratum was isolated less frequently and its incidence was less strongly or consistently correlated with weather or with the stage of lesion development. Fusarium spp. and R. solani were associated with dryer weather and higher temperatures. The Fusarium spp. were the fungi isolated most commonly during part of the dry season (January-March) and occurred more on early than on advanced lesions at one sample (January) when they may have been primary disease agents. R. solani was also more frequent on advanced lesions and may be principally a secondary coloniser of established lesions. The incidence of other fungi, which occurred less frequently and more sporadically, was not associated with weather or consistently with different stages of lesion development.