Population genomic footprints of host adaptation,introgression and recombination in coffee leaf rust

Coffee leaf rust, caused by Hemileia vastatrix (Hv), represents the biggest threat to coffee production worldwide and ranks amongst the most serious fungal diseases in history. Despite a recent series of outbreaks and emergence of hypervirulent strains, the population evolutionary history and potential of this pathogen remain poorly understood. To address this issue, we used restriction site‐associated DNA sequencing (RADseq) to generate ～19 000 single nucleotide polymorphisms (SNPs) across a worldwide collection of 37 Hv samples. Contrary to the long‐standing idea that Hv represents a genetically unstructured and cosmopolitan species, our results reveal the existence of a cryptic species complex with marked host tropism. Using phylogenetic and pathological data, we show that one of these lineages (C3) infects almost exclusively the most economically valuable coffee species (tetraploids that include Coffea arabica and interspecific hybrids), whereas the other lineages (C1 and C2) are severely maladapted to these hosts, but successfully infect diploid coffee species. Population dynamic analyses suggest that the C3 group may be a recent ‘domesticated’ lineage that emerged via host shift from diploid coffee hosts. We also found evidence of recombination occurring within this group, which could explain the high pace of pathotype emergence despite the low genetic variation. Moreover, genomic footprints of introgression between the C3 and C2 groups were discovered and raise the possibility that virulence factors may be quickly exchanged between groups with different pathogenic abilities. This work advances our understanding of the evolutionary strategies used by plant pathogens in agro‐ecosystems with direct and far‐reaching implications for disease control.     

Resistance inducers applied alone or in association with fungicide for the management of leaf rust and brown eye spot of coffee under field conditions

The use of resistance inducers is a promising development in the management of plant diseases, owing to their ability to control a broad spectrum of pathogens and improve the efficacy of fungicides. This study evaluates different sources of phosphonates (potassium, manganese, copper), a formulation prepared from the by‐products of the coffee industry (Greenforce CuCa), as well as the effects of their application, alone or in association with fungicide, in the management of two important coffee fungal diseases in Brazil: leaf rust and brown eye spot, caused by Hemileia vastatrix and Cercospora coffeicola, respectively. The effect of these products on defoliation, productivity and chemical composition of coffee beans (content of trigonelline, chlorogenic acid, caffeine and total soluble solids) was evaluated. Among all the alternative products tested individually, potassium phosphonate (P₂O₅—33.6% + K₂O—29%) stood out, particularly for rust control, which was similar to the results of fungicide treatments. Treatments with fungicide, Greenforce CuCa and cuprous oxide, individually, caused less plant defoliation. Regarding the chemical composition of the coffee beans, the manganese phosphonate treatment showed the highest values for trigonelline, chlorogenic acid, caffeine and total soluble solid content. The results of this study show that resistance inducers can be useful in disease management, may come to eventually replace traditional fungicides and can also contribute to the beverage quality.     

Diagrammatic Scale for Assessment of Bacterial Blight in Coffee Leaves

Due to the lack of a standardized visual method for assessing bacterial blight (Pseudomonas syringae pv. garcae) in coffee leaves, a diagrammatic scale was developed and validated to quantify the disease. Leaves were collected in crops and nursery with different intensity of symptoms, and the true severity was determined electronically. Based on the frequency distribution of severity values and according to the Weber–Fechner's law of visual stimulus, the minimum and maximum limits and the intermediate levels in the scale were determined. Validation was performed by ten evaluators who estimated the severity of 50 leaves with different intensity of symptoms. One evaluation was performed without diagrammatic scale and two evaluations with the scale at 7‐day intervals. The accuracy, precision, repeatability and reproducibility of the estimates were evaluated. The scale had nine levels: 0 (0%), 1 (0.1–0.99%), 2 (1–2%), 3 (2.01–4%), 4 (4.01–8%), 5 (8.01–16%), 6 (16.01–25%), 7 (25.01–45%) and 8 (≥45.1%). Using the scale, the evaluators were able to improve accuracy, precision, reproducibility and repeatability of estimates, compared to evaluators without scale. The scale was appropriate to visual estimation of severity of bacterial blight in coffee leaves.     

Development of pea bacterial blight caused by Pseudomonas syringae pv. pisi in winter and spring cultivars of combining peas (Pisum sativum) with different sowing dates

Pea bacterial blight occurred by natural infection in a field trial on peas in 1995. Disease development in the winter cultivars Rafale, Frilene and Froidure was compared with that in the spring cultivars Baccara, Conquest and Bohatyr, each sown on six dates in October, November, December, mid-March, late March and April. Disease incidence had reached 100% plants affected in all treatments by mid-July. Disease severity was greater in winter-sown (October, November or December) than in spring-sown peas of each cultivar at each assessment. Significant (P < 0.05) differences in disease severity occurred between cultivars in the winter-sown plots in May and June and the spring cultivars were affected more severely than the winter cultivars. Comparison of areas under the disease progress curves for both disease incidence and severity also showed that the winter-sown peas were more affected by disease than spring-sown peas and that spring cultivars were more severely affected than winter cultivars. Yield was strongly correlated with disease severity. A linear regression model suggested that, for peas sown in October, November or December, a yield loss of 0.5 tha^-1 occurred for each 10% increase in canopy area affected by pea bacterial blight.     

Foliar fertilizers for the management of phoma leaf spot on coffee seedlings

The growing demand for alternative strategies for plant disease management has sought a reduction in the use of fungicides via the employment of resistance inducers and foliar fertilizers, among others. Therefore, this study aimed at evaluating the following foliar fertilizers for the management of phoma leaf spot: a foliar fertilizer based on macro‐ and micronutrients (Fmm: 10% N, 4% S, B, 5% Fe and 5% Zn); one based on cobalt and molybdenum (Fcm: 2% Co and 3% Mo); manganese phosphite (FMn: 30% P₂O₅ and 9% Mn); and the FMn+Fmm, Fcm+Fmm and FMn+Fcm+Fmm associations compared to a boscalid fungicide and a control with no treatment. The disease severity, the chlorophyll a and b contents, the net photosynthetic rate (LPR), the phenylalanine ammonia lyase (PAL) activity and the lignin content in leaves were assessment. Based on the severity, the area under the disease severity progress curve (AUSPC) and the efficiency of disease control in relation to untreated plants were calculated. The experimental design was in randomized blocks with eight treatments and three replications. Treatments FMn+Fmm and Fcm were the most effective in reducing the AUPSC in comparison with the control and promoted an increased activity of PAL. FMn was the treatment that promoted the highest increase in the LPR. There were no effects of the treatments on the lignin content compared to the control.     

Time-concentration interplay in insecticide resistance among populations of the Neotropical coffee leaf miner,Leucoptera coffeella

1. Leucoptera coffeella is one of the main coffee pests in the Neotropical region and particularly important in crops cultivated under full sun, as in Brazil. The intensive use of insecticides in the last two decades, including molecules of recent use such as chlorantranilprole, has not been successful in suppressing this pest species.
2. Thus, study aimed to detect insecticide resistance and determine the levels of resistance to commercial formulations of the insecticides chlorantraniliprole, chlorpyrifos and thiamethoxam in populations of L. coffeella from Arabica coffee fields in western and the highlands of the state Bahia (Brazil). Furthermore, as the expression of insecticide toxicity varies with the length of exposure and the compound concentration, what is frequently neglected, these two variables were considered while determining the levels of resistance in nine populations.
3. Moderate levels of resistance were detected to chlorantranilprole in two populations, whereas the others exhibited low levels of resistance. All populations were resistant to thiamethoxam ranging from low to moderate levels. Low levels of resistance to chlorpyrifos were detected in all populations from western, but none from highlands region.
4. The western presents a more concerning scenario of insecticide resistance the L. coffeella, but the phenomenon requires attention in both regions.

     

Host specificity,pathogenicity and the presence of virulence genes in Iranian strains of Pseudomonas syringae pv. syringae from different hosts

Pseudomonas syringae pv. syringae (Pss) strains were isolated from almond, apricot, peach, pear, sweet cheery and wheat in Kohgiluye and Boyer-Ahmad, Kordestan, Fras and Chaharmahal and Bakhtiari provinces of Iran. The strains were examined for host specificity, the presence of virulence genes and pathogenicity on different hosts. After inoculation of isolates, in compatible reactions bacterial populations increased within six days of inoculation and final cell numbers increased several-fold over initial inoculum levels, but in incompatible reactions, bacterial populations declined within four days of inoculation. Almond, sweet cherry and wheat isolates induced progressive necrotic symptoms on almond leaves and stems. Apricot, peach and sweet cherry isolates induced necrotic lesions when inoculated on apricot leaves. On pear leaves and stems, only the pear isolate incited pathogenic reaction and isolates from other hosts did not. The syrB gene was detected in all of the tested isolates. Almond and pear isolates did not have the syrD gene. The sypA gene was detected in the almond, peach, pear and sweet cherry isolates while the sypB gene was detected in the apricot, peach, sweet cherry and wheat isolates. Almond, apricot, pear and wheat isolates gave negative results for the detection of nit gene. The gene Ach, was detected only in the peach isolate and gene hrmA, was detected only in the wheat isolate. This study indicates that host specificity exists among different Pss strains, and genes responsible for syringomycin and syringopeptin production contribute to the virulence of Pss strains.     

Evaluation of indigenous bacterial strains for biocontrol of the frogeye leaf spot of soya bean caused by Cercospora sojina

Aims: Assessment of biological control of Cercospora sojina, causal agent of frogeye leaf spot (FLS) of soya bean, using three indigenous bacterial strains, BNM297 (Pseudomonas fluorescens), BNM340 and BNM122 (Bacillus amyloliquefaciens). Methods and Results: From cultures of each bacterial strain, cell suspensions and cell‐free supernatants were obtained and assayed to determine their antifungal activity against C. sojina. Both mycelial growth and spore germination in vitro were more strongly inhibited by bacterial cell suspensions than by cell‐free supernatants. The Bacillus strains BNM122 and BNM340 inhibited the fungal growth to a similar degree (I～52–53%), while cells from P. fluorescens BNM297 caused a lesser reduction (I～32–34%) in the fungus colony diameter. The foliar application of the two Bacillus strains on soya bean seedlings, under greenhouse conditions, significantly reduced the disease severity with respect to control soya bean seedlings and those sprayed with BNM297. This last bacterial strain was not effective in controlling FLS in vivo. Conclusions: Our data demonstrate that the application of antagonistic bacteria may be a promising and environmentally friendly alternative to control the FLS of soya bean. Significance and Impact of the Study: To our knowledge, this is the first report of biological control of C. sojina by using native Bacillus strains.     

Macromolecules Released by a Plant Growth-promoting Rhizobacterium as Elicitors of Systemic Resistance in Tomato to Bacterial and Fungal Pathogens

One of 500 rhizobacteria isolated from soil, rhizosphere and rhizoplane of healthy tomato plants was previously selected in laboratory, greenhouse and field tests as a good inducer of systemic resistance. This plant growth‐promoting rhizobacterium (PGPR) was identified as Bacillus cereus by fatty‐acid analysis. Bacillus cereus bacterial cells were removed from liquid culture by centrifugation and the supernatant repeatedly dialyzed (cut‐off = 12 000 daltons) against distilled water. Dialysates applied to roots protected tomato plants against leaf fungal and bacterial pathogens, evidence that macromolecules synthesized by the PGPR and released into the environment act as elicitors of systemic resistance.     

Integrated biological control of bacterial speck and spot of tomato under field conditions using foliar biological control agents and plant growth-promoting rhizobacteria

Integration of foliar bacterial biological control agents and plant growth promoting rhizobacteria (PGPR) was investigated to determine whether biological control of bacterial speck of tomato, caused by Pseudomonas syringae pv. tomato, and bacterial spot of tomato, caused by Xanthomonas campestris pv. vesicatoria and Xanthomonas vesicatoria, could be improved. Three foliar biological control agents and two selected PGPR strains were employed in pairwise combinations. The foliar biological control agents had previously demonstrated moderate control of bacterial speck or bacterial spot when applied as foliar sprays. The PGPR strains were selected in this study based on their capacity to induce resistance against bacterial speck when applied as seed and soil treatments in the greenhouse. Field trials were conducted in Alabama, Florida, and California for evaluation of the efficacy in control of bacterial speck and in Alabama and Florida for control of bacterial spot. The foliar biological control agent P. syringae strain Cit7 was the most effective of the three foliar biological control agents, providing significant suppression of bacterial speck in all field trials and bacterial spot in two out of three field trials. When applied as a seed treatment and soil drench, PGPR strain Pseudomonas fluorescens 89B-61 significantly reduced foliar severity of bacterial speck in the field trial in California and in three of six disease ratings in the field trials in Alabama. PGPR strains 89B-61 and Bacillus pumilus SE34 both provided significant suppression of bacterial spot in the two field trials conducted in Alabama. Combined use of foliar biological control agent Cit7 and PGPR strain 89B-61 provided significant control of bacterial speck and spot of tomato in each trial. In one field trial, control was enhanced significantly with combined biological control agents compared to single agent inoculations. These results suggest that some PGPR strains may induce plant resistance under field conditions, providing effective suppression of bacterial speck and spot of tomato, and that there may be some benefit to the integration of rhizosphere-applied PGPR and foliar-applied biological control agents.     

Identification of a YAC clone carrying the Xa-1 allele, a bacterial blight resistance gene in rice

Map-based cloning methods have been applied for isolation of Xa-1, one of the bacterial blight resistance genes in rice.Xa-1 was previously mapped on chromosome 4 using molecular markers. For positional cloning of Xa-1, a high-resolution genetic map was made for theXa-1 region using an F₂ population of 402 plants and additional molecular markers. Three restriction fragment length polymorphism (RFLP) markers, XNpb235, XNpb264 and C600 were found to be linked tightly to Xa-1, with no recombinants, and U08 ₇₅₀ was mapped 1.5 cM from Xa-1. The screening of a yeast artificial chromosome (YAC) library using theseXa-1-linked RFLP markers resulted in the identification of ten contiguous YAC clones. Among these, one YAC clone, designated Y5212, with an insert of 340 kb, hybridized with all three tightly linked markers. This YAC was confirmed to possess the Xa-1 allele by mapping the Xa-1 gene between both end clones of this YAC (Y5212R and Y5212L).     

转拟南芥AtNPR1基因增强水稻对水稻白叶枯病和稻瘟病的抗性

AtNPR1基因是拟南芥系统获得抗性的一个重要调节基因,在拟南芥中过量表达AtNPR1基因能使拟南芥对细菌和真菌的抗性同时增强.为了研究在水稻中过量表达AtNPR1基因对水稻抗病性的影响,将该基因转入到广西主栽籼稻恢复系品种桂99中.经PCR验证得到了79株转基因植株,DNA斑点杂交表明ATNPR1基因已经整合到桂99染色体DNA中.Northern杂交和RT-PCR分析表明,AtNPR1基因在桂99中已经表达;同时还检测了转基因植株对水稻白叶枯病和稻瘟病的抗性,结果表明转基因植株对该两种病害的抗性均显著增强.     

Effect of relative humidity,temperature and fungicide on germination of conidia of Cercospora canescens caused the Cercospora leaf spot disease in mungbean

The aim of the present investigation was to determine the impact of relative humidity (RH) and temperature on conidial germination, nuclear position and effect of important fungicides on growth and conidial germination of Cercospora canescens. Germination of conidia was observed at RH range 92–100% at 5–35°C. Significant interaction between temperature and RH indicated that higher humidity and high temperature promoted quick germination both in the presence and absence of free moisture. Although in absence of free moisture at 92–95% RH higher temperatures 25–35°C promoted quick evaporation of moisture and no conidial germination. Number of germtube was increased significantly at the optimum temperature 25–30°C and higher humidity (98–100%). But higher temperature 25–35°C with lower RH did not support the conidial germination. This finding is very important for disease forecasting using meteorological data. The spray of Carbendazim as contact fungicide may not be useful since it is not effective against the conidia of C. canescens. Triadimefon did not inhibit the conidia germination but completely inhibited mycelium development at 50 μg/ml. Propriconazole inhibited both conidia germination and mycelial development. Therefore, Propiconazole may be taken as protective as well as curative spray. In non-systemic fungicide, Copper oxychloride gave anticipated result by inhibiting both conidial germination and mycelium development. Therefore, copper oxychloride can be used as protectant fungicides for Cercospora leaf spot caused by C. canescens.     

Exogenous N‐acyl‐homoserine lactones enhance the expression of flagella of Pseudomonas syringae and activate defence responses in plants

In order to cope with pathogens, plants have evolved sophisticated mechanisms to sense pathogenic attacks and to induce defence responses. The N‐acyl‐homoserine lactone (AHL)‐mediated quorum sensing in bacteria regulates diverse physiological processes, including those involved in pathogenicity. In this work, we study the interactions between AHL‐producing transgenic tobacco plants and Pseudomonas syringae pv. tabaci 11528 (P. syringae 11528). Both a reduced incidence of disease and decrease in the growth of P. syringae 11528 were observed in AHL‐producing plants compared with wild‐type plants. The present data indicate that plant‐produced AHLs enhance disease resistance against this pathogen. Subsequent RNA‐sequencing analysis showed that the exogenous addition of AHLs up‐regulated the expression of P. syringae 11528 genes for flagella production. Expression levels of plant defence genes in AHL‐producing and wild‐type plants were determined by quantitative real‐time polymerase chain reaction. These data showed that plant‐produced AHLs activated a wide spectrum of defence responses in plants following inoculation, including the oxidative burst, hypersensitive response, cell wall strengthening, and the production of certain metabolites. These results demonstrate that exogenous AHLs alter the gene expression patterns of pathogens, and plant‐produced AHLs either directly or indirectly enhance plant local immunity during the early stage of plant infection.