Flower infection of Brassica oleracea with Xanthomonas campestris pv. campestris results in high levels of seed infection

During seed production, Brassica seed may become infected with Xanthomonas campestris pv. campestris after systemic colonization of plants upon leaf infection, or alternatively, after flower infection. Polytunnel experiments were conducted in 2007 and 2008 to study the relative importance of these colonization routes resulting in seed infection. Cauliflower plants (Brassica oleracea) were spray-inoculated at the 8-leaf stage, after formation of cauliflowers or during flowering, at which stage leaves or blossoms were inoculated. Inoculation at all stages resulted in a relatively high percentage of systemic infection; the average estimated infection incidences for stem base and peduncle infections were 16 % and 19 %, respectively. When seed samples were examined by dilution plating for deep-seated infection following hot water treatment, Xcc was detected in 61 % of the 23 seed samples harvested from plants with inoculated flowers. However, symptom development in seedlings raised from the seeds could not be confirmed in a grow-out test under favourable conditions for Xcc infection at a high RH (>95 %) and a relative high temperature (28 °C). Xcc was not detected in 59 seed samples harvested from leaf-inoculated plants with the exception of one sample from plants inoculated at peduncle formation. In a third polytunnel experiment carried out in 2009, the population dynamics of Xcc on inoculated flowers was investigated. Following spray-inoculation of flowers, 52 % of the flowers were infected with Xcc. During development of siliques, infection incidence decreased slowly and at 56 dpi, 20 % of the superficially disinfected siliques were infected with Xcc. It was estimated that 0.18 % of the seeds was infected and that 1–10 % of the infected siliques contained infected seeds. The implications of these results for control of Xcc in a seed production crop are discussed.     

Molecular epidemiology of cassava mosaic disease in Madagascar

Cassava is the staple food for hundreds of millions of people in Africa but its cultivation is seriously constrained by cassava mosaic disease (CMD) in Madagascar, and in Africa in general. This study identified the cassava mosaic geminiviruses (CMGs) involved in CMD in Madagascar and their associated epidemiological characteristics from countrywide surveys. Molecular characterization of CMGs in Madagascar revealed an unprecedented diversity and co‐occurrence of six viruses: African cassava mosaic virus (ACMV), East African cassava mosaic Cameroon virus (EACMCV), East African cassava mosaic Kenya virus (EACMKV), East African cassava mosaic virus (EACMV), South African cassava mosaic virus (SACMV) and the recently described Cassava mosaic Madagascar virus (CMMGV). Distinct geographical distributions were observed for the six viruses. While ACMV was more prevalent in the central highlands, EACMV and EACMKV were prevalent in lowlands and coastal regions. Both EACMCV and SACMV occurred in almost all the localities visited. PCR diagnosis revealed that mixed infection (up to four co‐infected viruses) occurred in 21% of the samples and were associated with higher symptom severity scores. Pairwise comparisons of virus associations showed that EACMCV was found in mixed infections more often than expected while ACMV and SACMV were mostly found in single infections. A greater abundance of whiteflies was observed in lowland and coastal areas. Nevertheless, infected cuttings remain the primary source of CMD propagation (95%) in Madagascar.     

基于链特异性RNA-seq的禾谷镰刀菌全生活史转录组分析

为明确植物病原真菌禾谷镰刀菌Fusarium graminearum全生活史的转录组特征和基因表达模式,采用生物信息学技术对其生活史不同阶段15个时期或组织的链特异性RNA-seq数据进行分析。结果表明,共有8 106个基因在所有时期均有表达,为禾谷镰刀菌生活史核心基因,占总基因数的47.2%;有性生殖和侵染过程中表达的基因数相对较多,其中有性生殖后期表达的基因数最多,达15 221个;无性产孢和侵染小麦穗过程中基因表达水平整体较高,而分生孢子中基因表达水平最低。在燕麦培养基上禾谷镰刀菌气生菌丝的基因表达模式与侵染过程中的基因表达模式较为相似,而与营养生长菌丝的基因表达模式差异较大。该菌次生代谢物合成特征酶基因和分泌蛋白基因的表达模式均分成3类,即分别在侵染、营养生长和有性生殖过程上调表达,暗示其在生活史不同阶段的特异性功能。     

Generation and evaluation of movement protein‐specific single‐chain antibodies for delaying symptoms of Tomato spotted wilt virus infection in tobacco

This study investigated whether single‐chain antibodies (scFvs) specific for a viral movement protein could accumulate in the plant cell cytosol and restrict viral systemic infection in plants. Nine chicken scFv fragments against the Tomato spotted wilt virus (TSWV) movement protein (NS_M) were isolated by phage display. Soluble scFvs were produced in bacteria and the NS_M binding activity of purified scFvs was confirmed. The nine scFv genes were cloned into a plant expression vector enabling recombinant protein accumulation in the plant cell cytosol. Immunoblot analysis demonstrated that two of the nine chicken scFvs accumulated to high levels (5·9 and 8·0% of total soluble protein). Bioassays of viral infection using transgenic tobacco plants producing NS_M‐specific chicken scFvs showed delayed symptom development when compared to non‐transgenic control plants, indicating that expression of antibodies recognizing the TSWV movement protein is a potential strategy for generating resistant plants.     

Cassava common mosaic virus infection causes alterations in chloroplast ultrastructure,function, and carbohydrate metabolism of cassava plants

Cassava common mosaic virus (CsCMV) is a potexvirus that causes systemic infections in cassava plants, leading to chlorotic mosaic and producing significant yield losses. To date, the physiological alterations and the mechanism underlying biotic stress during the cassava–CsCMV compatible interaction remains unknown. In this study, we found that CsCMV infection adversely modified chloroplast structure and had functional effects on chloroplasts in source leaves during the course of viral infection. Extrusion of the chloroplast membrane with amoeboid-shaped appearance and disorganized grana stacks were observed in infected mesophyll cells. These alterations were associated with up to 35% reduction of relative chlorophyll content, and a decline of CO₂ fixation (13.5% and 24.2% at 90 and 210 days after planting, respectively). The effects of CsCMV infection on the performance index on absorption basis dropped up to 37%. The analysis of chlorophyll a fluorescence showed a progressive loss of both oxygen evolving complex activity and “connectivity” within the tripartite system (core antenna-LHCII-reaction centre). Here, we report the latter phenomenon for the first time in a viral infection. The oxidative stress process was observed in CsCMV-infected plants (20.8% reduction of antioxidant capacity with respect to noninfected plants). Other effects of the pathogen included reduction of starch and maltose content in source leaves, and a significant increase (24.7%) of the sucrose:starch ratio, which indicates an altered pattern of carbon allocation. Our results suggest that CsCMV induces chloroplast distortion associated with progressive chloroplast function loss and diversion of carbon flux in source leaf tissue, leading to the loss of cassava tuber yield.     

Tomato yellow leaf curl virus effects on chloroplast biogenesis and cellular structure

Tomato yellow leaf curl virus (TYLCV) is a begomovirus causing significant crop losses in tomatoes worldwide. TYLCV infection has several defined symptoms that are characterized at the whole plant level that include yellowing and curling of the leaves, stunting and growth retardation, and severe yield losses that can reach 100%. While the etiology and phenotypic symptoms of TYLCV infection were characterized, little is known about the cellular basis of these symptoms. Our goal in the present study is to describe some of the microscopic features of the disease while comparing TYLCV-susceptible and resistant near-isogenic tomato plants under infection. We depict the cellular changes that occur in susceptible line as a result of TYLCV infection that include: epidermal cell sizes reduction, accompanied with modifications in cell shape; reduction in plastid number, plastid size and chlorophyll content; diminished expression genes encoding LHC, Rub L, CAB4, and CAB8 and a surge in plasmodesmata number after infection. We conclude that TYLCV affect many targets or that it has a very critical target in cellular development.     

Inhibition on brown rot disease and induction of defence response in harvested peach fruit by nitric oxide solution

Nitric oxide (NO) is an important signal molecule involved in numerous plant responses to biotic and abiotic stresses. The effect of nitric oxide (NO) solution on pathogen infection and defence response of peach (Prunus persica (L.) Batsch) fruit against brown rot disease caused by Monilinia fructicola was investigated. The results showed that 15 μmol l^?1 NO solution did not significantly inhibit spore germination, germ tube length or pathogenicity of M. fructicola, but significantly reduced disease incidence and lesion areas in the fruit. Although 100 μmol l^?1 NO solution effectively inhibited the spore germination, germ tube elongation and pathogenicity of M. fructicola, the high concentration of NO solution caused damage to the fruit. Moreover, 15 μmol l^?1 NO enhanced the activities of chitinase (CHI) and β-1,3-glucanase (GNS) in the fruit. RT-PCR analysis showed that the expression of four genes, CHI, GNS, pathogenesis-related protein 1 and 10 genes (PR-1, PR-10) all increased after NO treatment. Conversely, pretreatment with 100 μmol l^?1 NO scavenger, 2-4-carboxyphenyl-4,4,5,5- tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), rendered the fruit relatively susceptible to pathogen infection and inhibited the defence response of the fruit. These results suggest that NO solution treatment can protect peach fruit from pathogen infection by inducing the activities of the defence enzymes and the expression of PR genes.     

The role of seed infection level and fungicide seed treatments in control of net blotch in barley

The effect of seed-borne net blotch infection in barley on the agronomic characters yield, thousand kernel weight, grain volume weight, protein, starch and plant density was investigated in field trials using six net blotch infection levels (100/0, 80/20, 60/40, 40/60, 20/80 and 0/100 % high/low infected seed) and four fungicide seed treatments. The untreated trials showed significant differences between infection levels only for some agronomic characters, years and levels. Seed treatment with Anchor (carboxin + thiram) reduced net blotch by nearly 100 %, compared to 81 % by Fungazil A 25 (imazalil), 60 % by Rancona i-MIX (ipconazole + imazalil) and 54 % by Rubin TT (triticonazole + prochloraz + pyrimethanil). Anchor significantly increased yield 3–7 %, thousand kernel weight 3–13 %, grain volume weight 3–9 % and protein content 1–7 % in the first year, but not in the second. Fungazil A 25 had a low positive effect on agronomic characters, while Rancona i-MIX and Rubin TT had a positive, negative or no effect. The correlation between percent infected kernels in the conventional osmotic method and real-time PCR results was high, showing a potential for complementing traditional plate test methods with molecular methods that needs further optimization. The development of net blotch and its effect on agronomic characters thus depended on many other factors than the seed infection level. The current recommendations for seed treatment could be considered a preventative measure.     

Screening for Differential Gene Expression in Atropa belladonna Leafy Gall Induced Following Rhodococcus fascians Infection

To better characterise at the molecular level the nature of plant responses to infection by Rhodococcus fascians PCR-based differential display patterns of Atropa belladonna leafy gall (LG) and non-infected plant tissues were compared. Six differentially expressed genes were identified and their altered expression was confirmed by RT-PCR. Three of them corresponded to up-regulated genes which encode proteins involved in plant defence. The three remaining cDNA fragments which correspond to down-regulated genes in LG, encoded proteins with similarity to a multicystatin, a miraculin and a methallothionein-like protein, respectively. Upon elimination of the bacteria from infected plant tissue, the expression of up-regulated genes was maintained, whereas expression of down-regulated genes resumed suggesting a potential role of these up-regulated genes in plant growth and development.     

Soilborne viruses: advances in virus movement,virus induced gene silencing,and engineered resistance

Until recently soilborne plant viruses were considered important only because they are causative agents for agricultural diseases. In recent years, soilborne plant viruses have played a significant role in advancing research into mechanisms of plasmodesmata transport, gene silencing, and engineered resistance to plant pathogens. Three different mechanisms by which viruses move through plasmodesmata have been identified using dianthoviruses, nepoviruses, and benyviruses. The infectious clone of the tobravirus Tobacco rattle virus (TRV) has become an important tool for studying virus induced gene silencing and may be a tool to silence meristematic gene expression. Investigations of soilborne viruses may enable the development of new strategies for control of soilborne diseases. The potential use of pathogen-derived resistance to control soilborne viruses is currently being explored in several laboratories.     

Subcelullar localization of proteins associated with <Emphasis Type="Italic">Prune dwarf virus</Emphasis> replication

The fungus Colletotrichum lindemuthianum is the causal agent of anthracnose, one of the most severe diseases of the common bean (Phaseolus vulgaris). The infection process begins with the adhesion of conidia to the plant’s surface. Appressoria are then formed, allowing penetration of the fungus. Next, the biotrophic phase begins, followed by the necrotrophic phase. Due to the peculiar nutrition mode of the fungus, including both of the previously mentioned stages, it is of great interest to determine which genes are involved in the transition between the two phases during the infection process. To determine this, suppression subtractive hybridization (SSH) was used in association with qRT-PCR in the present study. These methods allowed for the identification of genes that were differentially expressed at each developmental stage of the fungus in the plant. This is the first report on the use of the cited techniques to evaluate the infectious cycle of the fungus. A total of 175 sequences exhibited significant identity (e?≤?10^?5) with sequences present in the sequenced genomes of P. vulgaris and C. lindemuthianum; approximately 41 % of those were determined to belong to the fungus, and 59 % were determined to belong to the plant. Of the predicted sequences, 68 % were of unknown function or were not found in the databases. Among the analyzed expressed sequence tags (ESTs), sequences were found that encode proteins related to: primary and secondary metabolism; the transport of different compounds; the degradation/modification of proteins; cell regulation and signaling; cellular stress response; and the degradation of exogenous compounds. The obtained results allowed for the identification of sequences encoding proteins that are essential for the progression of anthracnose. Furthermore, it was possible to identify new genes, the functions of which have not yet been described, and even to identify unique genes of C. lindemuthianum that are involved in the pathogenicity and virulence of this fungus.     

An antibiotic fusaricidin: a cyclic depsipeptide from Paenibacillus polymyxa E681 induces systemic resistance against Phytophthora blight of red-pepper

In this study fusaricidin, a cyclic depsipeptide isolated from Paenibacillus polymyxa E681 (E681), was demonstrated to control Phytophthora blight infection caused by Phytophthora capsici in red-pepper. The minimal inhibitory concentration (MIC) of fusaricidin was found to be 16 ppm against P. capsici. The disease severity of P. capsici was 40% at 0.1 ppm of fusaricidin when compared with water-treated control (81.7%) on post-treatment, whereas the disease severities on pre-treatment were 45% and 83.3% in fusaricidin (0.1 ppm) and water-treated control, respectively, in red-pepper plants. Significant (P?<?0.05) disease suppression was observed on treatment with fusaricidin (0.1 ppm) by foliar spray and soil drench. The disease severity was drastically reduced to 3.3% by soil drench of fusaricidin (1.0 ppm), whereas in water-treated control, the disease severity was 83.3% in the first experiment. Fusaricidin at 0.1 ppm reduced disease severity of P. capsici to 27.5% when compared with positive control (43.1%) and water-treated control (66.2%) in the second experiment. Soft rot disease in tobacco was suppressed upon treatment with fusaricidin at 1.0 ppm by leaf infiltration. RT-PCR analyses of Arabidopsis thaliana revealed that there was an up-regulation of pathogenesis-related (PR) gene expression in wild type A. thaliana (Col-0), while there was no accumulation of PR genes, which implies that the mechanism of protection might be based on a salicylic acid-dependent pathway. This is the first report that fusaricidin exhibits protection against plant pathogens in addition to activity as an antibiotic agent. Hence, E681 can play a role in plant protection by secretion of ISR elicitors including fusaricidin.     

Health of farmer-saved maize seed in north-east Nigeria

Many Nigerian farmers depend for their seed on seed-producing farmers, the so-called informal Seed System (SS), but seed quality of the SS is unknown. Farmers planting low quality seed risk poor field emergence and low plant vigour as a result of low physiological quality or infection with seed-borne pathogens. The objective of this research was to test seed quality of maize seed from the informal SS in north-east Nigeria. A total of 46,500 seeds (93 samples of 500 seeds each) were tested for germination, off-types and seed health. Seed pathology was quantified by plating disinfected seeds onto agar, and identifying the fungi present after 3 days incubation. Twelve seed-borne pathogens were identified including Bipolaris maydis (found in 45 % of the farmer-produced samples), Botryodiplodia theobromae (97 %) and Curvularia lunata (38 %). All samples were infected with Fusarium verticillioides, with a median infection incidence of 59 % (2009) and 51 % (2010). None of the 93 samples tested passed the demands for certified seed of the National Agricultural Seeds Council (NASC) in Nigeria, in particular the maximum limit of five off-types per kg seed sample. Based on these results, seed-producing farmers must improve the health of seed. The NASC should revise the standards for off-type seeds to minimize the time spent by farmers sorting planting material.     

Increased resistance to fusarium wilt in transgenic tobacco lines co‐expressing chitinase and wasabi defensin genes

Marker‐free transgenic tobacco (Nicotiana tabacum) lines containing a chitinase (ChiC) gene isolated from Streptomyces griseus strain HUT 6037 were produced by Agrobacterium‐mediated transformation. One marker‐free transgenic line, TC‐1, was retransformed with the wasabi defensin (WD) gene, isolated from Wasabia japonica. Of the retransformed shoots, 37% co‐expressed the ChiC/WD genes, as confirmed by western and northern analyses. Southern blot analysis showed that no chromosomal rearrangement was introduced between the first and the second transformation. Transgenic lines either expressing ChiC or WD, or co‐expressing both genes were challenged with Fusarium oxysporum f.sp. nicotianae (Fon). Assessment of in vitro plant survival in the presence of Fon showed that transgenic lines co‐expressing both genes had significantly enhanced protection against the fungus (infection indices 0·0–1.·2) compared with corresponding isogenic lines expressing either of the genes (infection indices 2·5–9·8). Whole‐plant infection indices in transgenic lines were significantly related (r = 0·93, P < 0·01) to the extent of root colonization of the host, which ranged from 2·1% to 11·3% in lines co‐expressing both genes, and from 16·8% to 37·7% in lines expressing just one of the genes (compared with 86·4% in non‐transformed controls). Leaf extracts of transgenic lines also inhibited mycelial growth of Fon in vitro and caused hyphal abnormalities.     

Cellulysin induces downy mildew disease resistance in pearl millet driven through defense response

The responses to cellulysin as an immune inducer in pearl millet that confers downy mildew resistance mediated through lipoxygenase (LOX), a jasmonate-dependent enzyme involved in defence signalling, are discussed in this paper. The susceptible pearl millet cultivar 7042S was treated with cellulysin at 10, 15, 20, 30 and 50 μg/ml concentrations. All tested concentrations showed enhanced seed germination and seedling vigour when compared with the untreated control. Maximum seed germination of 92 % and seedling vigour was obtained following 20 μg/ml cellulysin treatment. Significant (P?<?0.05) downy mildew disease protection of 67 % and 71 % was observed when cellulysin was used at 20 μg/ml under greenhouse and field conditions, respectively. Further studies showed that the resistance induced by cellulysin treatment in pearl millet plant was systemic, required a minimum of 4 days to achieve maximum resistance development after pathogen inoculation seedling inoculation (five-day-old), and was sustained throughout the plant’s life. Plants raised from cellulysin-treated seeds and challenge inoculated at tillering (25-day-old) and inflorescence (45-day-old) showed persistence in resistance till the end of the crop period. A notable increase in LOX activity was observed in all the tested concentrations of cellulysin in plants inoculated with the pathogen at 24 h, compared to the control. However, a maximum 6-fold increase in LOX activity was noticed using a cellulysin concentration of 20 μg/ml 48 hours post inoculation. In contrast, glucanase (GLU) activity was high in control inoculated seedlings, but was low in cellulysin treated samples at all time intervals. The optimal cellulysin treatment (20 μg/ml) provided enhanced vegetative and reproductive parameters that resulted in higher yield compared to the untreated control.