Common bean (Phaseolus vulgaris L.) is an important legume with high nutritional value. In Nicaragua, certified healthy seeds of local bean varieties are not available, and seedborne fungi have ...gained little attention. Here, were surveyed seedborne pathogenic fungi in an important local bean cultivar, 'INTA Rojo'. Beans grown in the four main production areas in Nicaragua (Boaco, Carazo, Estelí, Matagalpa) for future use as seed stock were sampled from four seed storehouses and six seed lots. A total of 133 fungal strains were isolated from surface-sterilized beans and inoculated to healthy lima beans (Phaseolus lunatus) under controlled conditions. Eighty-seven isolates caused symptoms of varying severity in the seedlings, including discoloration, necrotic lesions, cankers, rot, and lethal necrosis. Pathogenic isolates were divided into eight phenotypically distinguishable groups based on morphology and growth characteristics on artificial growth medium, and further identified by analysis of the internal transcribed spacer sequences (ITS1 and ITS2) of the ribosomal RNA genes. The pathogenic isolates belonged to eight genera. Fusarium spp. (F. chlamydosporum, F. equiseti, F. incarnatum), Lasiodiplodia theobromae, Macrophomina phaseolina, and Penicillium citrinum were the most damaging and common fungi found in the seed lots. Furthermore, Corynespora cassiicola, Colletotrichum capsisi, Colletotrichum gloeosporioides, Aspergillus flavus, and Diaporthe sp. (Phomopsis) were seedborne in cultivar 'INTA Rojo' and found to be pathogenic to bean seedlings. This study reveals, for the first time, many seedborne pathogenic fungi in beans in Nicaragua; furthermore, prior to this study, little information was available concerning F. equiseti, F. incarnatum, L. theobromae, C. cassiicola, and Diaporthe spp. as seedborne pathogens of common bean. Our results lay the basis for developing diagnostic tools for seed health inspection and for further study of the epidemiology, ecology, and control of the pathogenic fungi of common beans in the field.
Virus diseases caused by co-infection with
(SPFMV) and
(SPCSV) are a severe problem in the production of sweetpotato (
L.). Traditional molecular virus detection methods include nucleic acid-based ...and serological tests. In this study, we aimed to validate the use of a non-destructive imaging-based plant phenotype platform to study plant-virus synergism in sweetpotato by comparing four virus treatments with two healthy controls.
By monitoring physiological and morphological effects of viral infection in sweetpotato over 29 days, we quantified photosynthetic performance from chlorophyll fluorescence (ChlF) imaging and leaf thermography from thermal infrared (TIR) imaging among sweetpotatoes. Moreover, the differences among different treatments observed from ChlF and TIR imaging were related to virus accumulation and distribution in sweetpotato. These findings were further validated at the molecular level by related gene expression in both photosynthesis and carbon fixation pathways.
Our study validated for the first time the use of ChlF- and TIR-based imaging systems to distinguish the severity of virus diseases related to SPFMV and SPCSV in sweetpotato. In addition, we demonstrated that the operating efficiency of PSII and photochemical quenching were the most sensitive parameters for the quantification of virus effects compared with maximum quantum efficiency, non-photochemical quenching, and leaf temperature.
Rhizophagus irregularis, an arbuscular mycorrhizal fungus, and Bacillus amyloliquefaciens, a bacterium, are microorganisms that promote plant growth. They associate with plant roots and facilitate ...nutrient absorption by their hosts, increase resistance against pathogens and pests, and regulate plant growth through phytohormones. In this study, eight local plant species in Finland (Antennaria dioica, Campanula rotundifolia, Fragaria vesca, Geranium sanguineum, Lotus corniculatus, Thymus serpyllum, Trifolium repens, and Viola tricolor) were inoculated with R. irregularis and/or B. amyloliquefaciens in autoclaved substrates to evaluate the plant growth-promoting effects of different plant/microbe combinations under controlled conditions. The eight plant species were inoculated with R. irregularis, B. amyloliquefaciens, or both microbes or were not inoculated as a control. The impact of the microbes on the plants was evaluated by measuring dry shoot weight, colonization rate by the arbuscular mycorrhizal fungus, bacterial population density, and chlorophyll fluorescence using a plant phenotyping facility. Under dual inoculation conditions, B. amyloliquefaciens acted as a "mycorrhiza helper bacterium" to facilitate arbuscular mycorrhizal fungus colonization in all tested plants. In contrast, R. irregularis did not demonstrate reciprocal facilitation of the population density of B. amyloliquefaciens. Dual inoculation with B. amyloliquefaciens and R. irregularis resulted in the greatest increase in shoot weight and photosynthetic efficiency in T. repens and F. vesca.
Plants recognize unrelated viruses by the antiviral defense system called RNA interference (RNAi). RNAi processes double-stranded viral RNA into small RNAs (sRNAs) of 21-24 nucleotides, the ...reassembly of which into longer strands
allows virus identification by comparison with the sequences available in databases. The aim of this study was to compare the virus detection sensitivity of sRNA-based virus diagnosis with the established virus species-specific polymerase chain reaction (PCR) approach. Viruses propagated in tobacco plants included three engineered, infectious clones of
(PVA), each carrying a different marker gene, and an infectious clone of
(PVY). Total RNA (containing sRNA) was isolated and subjected to reverse-transcription real-time PCR (RT-RT-PCR) and sRNA deep-sequencing at different concentrations. RNA extracted from various crop plants was included in the reactions to normalize RNA concentrations. Targeted detection of selected viruses showed a similar threshold for the sRNA and reverse-transcription quantitative PCR (RT-qPCR) analyses. The detection limit for PVY and PVA by RT-qPCR in this study was 3 and 1.5 fg of viral RNA, respectively, in 50 ng of total RNA per PCR reaction. When knowledge was available about the viruses likely present in the samples, sRNA-based virus detection was 10 times more sensitive than RT-RT-PCR. The advantage of sRNA analysis is the detection of all tested viruses without the need for virus-specific primers or probes.
Viruses infecting wild flora may have a significant negative impact on nearby crops, and vice-versa. Only limited information is available on wild species able to host economically important viruses ...that infect sweetpotatoes (Ipomoea batatas). In this study, Sweet potato chlorotic fleck virus (SPCFV; Carlavirus, Betaflexiviridae) and Sweet potato chlorotic stunt virus (SPCSV; Crinivirus, Closteroviridae) were surveyed in wild plants of family Convolvulaceae (genera Astripomoea, Ipomoea, Hewittia and Lepistemon) in Uganda. Plants belonging to 26 wild species, including annuals, biannuals and perennials from four agro-ecological zones, were observed for virus-like symptoms in 2004 and 2007 and sampled for virus testing. SPCFV was detected in 84 (2.9%) of 2864 plants tested from 17 species. SPCSV was detected in 66 (5.4%) of the 1224 plants from 12 species sampled in 2007. Some SPCSV-infected plants were also infected with Sweet potato feathery mottle virus (SPFMV; Potyvirus, Potyviridae; 1.3%), Sweet potato mild mottle virus (SPMMV; Ipomovirus, Potyviridae; 0.5%) or both (0.4%), but none of these three viruses were detected in SPCFV-infected plants. Co-infection of SPFMV with SPMMV was detected in 1.2% of plants sampled. Virus-like symptoms were observed in 367 wild plants (12.8%), of which 42 plants (11.4%) were negative for the viruses tested. Almost all (92.4%) the 419 sweetpotato plants sampled from fields close to the tested wild plants displayed virus-like symptoms, and 87.1% were infected with one or more of the four viruses. Phylogenetic and evolutionary analyses of the 3'-proximal genomic region of SPCFV, including the silencing suppressor (NaBP)- and coat protein (CP)-coding regions implicated strong purifying selection on the CP and NaBP, and that the SPCFV strains from East Africa are distinguishable from those from other continents. However, the strains from wild species and sweetpotato were indistinguishable, suggesting reciprocal movement of SPCFV between wild and cultivated Convolvulaceae plants in the field.
Recent advances in high-throughput sequencing technologies and bioinformatics have generated huge new opportunities for discovering and diagnosing plant viruses and viroids. Plant virology has ...undoubtedly benefited from these new methodologies, but at the same time, faces now substantial bottlenecks, namely the biological characterization of the newly discovered viruses and the analysis of their impact at the biosecurity, commercial, regulatory, and scientific levels. This paper proposes a scaled and progressive scientific framework for efficient biological characterization and risk assessment when a previously known or a new plant virus is detected by next generation sequencing (NGS) technologies. Four case studies are also presented to illustrate the need for such a framework, and to discuss the scenarios.
In seed plants, strigolactones (SLs) regulate architecture and induce mycorrhizal symbiosis in response to environmental cues. SLs are formed by combined activity of the carotenoid cleavage ...dioxygenases (CCDs) 7 and 8 from 9-cis-β-carotene, leading to carlactone that is converted by cytochromes P450 (clade 711; MAX1 in Arabidopsis) into various SLs. As Physcomitrella patens possesses CCD7 and CCD8 homologs but lacks MAX1, we investigated if PpCCD7 together with PpCCD8 form carlactone and how deletion of these enzymes influences growth and interactions with the environment.
We investigated the enzymatic activity of PpCCD7 and PpCCD8 in vitro, identified the formed products by high performance liquid chromatography (HPLC) and LC-MS, and generated and analysed ΔCCD7 and ΔCCD8 mutants.
We defined enzymatic activity of PpCCD7 as a stereospecific 9-cis-CCD and PpCCD8 as a carlactone synthase. ΔCCD7 and ΔCCD8 lines showed enhanced caulonema growth, which was revertible by adding the SL analogue GR24 or carlactone. Wild-type (WT) exudates induced seed germination in Orobanche ramosa. This activity was increased upon phosphate starvation and abolished in exudates of both mutants. Furthermore, both mutants showed increased susceptibility to phytopathogenic fungi.
Our study reveals the deep evolutionary conservation of SL biosynthesis, SL function, and its regulation by biotic and abiotic cues.
Viral genome-linked protein (VPg) of potyviruses is involved in multiple steps of the potyvirus infection cycle, including viral multiplication and movement in plants. Recently, we showed that VPg of ...Potato virus A (PVA; genus Potyvirus) suppresses sense-mediated RNA silencing, which is linked to one or both nuclear or nucleolar localization. Here, we studied interactions between VPg and components of the plant RNA silencing pathway. Results showed that VPg interacts with the SGS3 protein of Solanum tuberosum and Arabidopsis thaliana, as shown by yeast two-hybrid analysis and bimolecular fluorescence complementation assays. VPg-SGS3 interactions co-localized with small cytoplasmic bodies that contained plant RNA-dependent RNA polymerase 6 (RDR6) (likely SGS3/RDR6 bodies). The N-terminal zinc finger (ZF) domain of SGS3 was the main determinant of the VPg interaction. Our data also suggest that the ZF domain controls SGS3 localization. SGS3 homodimerization was controlled by multiple protein regions. The VPg-SGS3 interaction appeared beneficial for PVA, as viral RNA levels correlated positively with sgs3 mRNA levels in the SGS3-silenced and SGS3-overexpressing leaves of Nicotiana benthamiana. The data support the idea that VPg acts as a suppressor of RNA silencing and suggest that an interaction with SGS3 may be important, especially in suppression of sense-mediated RNA silencing.
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