Sustainable control of pea bacterial blight : approaches for durable genetic resistance and biocontrol by endophytic bacteria

Key-words: bacterial blight, biological control, biodiversity, endophytic bacteria, L-form, pea, PDRl retrotransposon, Pisum sativum, Pisum abyssinicum, Pseudomonas syringae pv. pisi, race specific resistance, race non-specific resistance, Spanish landraces.Pea bacterial blight (Pseudomonas syringae pv. pisi) occurs worldwide and can cause severe damage under cool and wet conditions particularly at the seedling stage in wintersown crops. Seven Ps. syr. pv. pisi races are currently recognized. There are no resistant cultivars to race 6, which is becoming increasingly important. Current disease control measures include disease avoidance through seed testing and the deployntent of resistant cultivars with race specific resistance gene(s). In the present study two novel control measures were investigated with the potential for integration to give a durable and sustainable disease control. The first was breeding for resistance based on race non-specific resistance present in Pisum abyssinicum, which confers resistance to all races, including race 6. Its mode of inheritance was investigated through a crossing programme with Pisum sativum cultivars. Resistance was controlled by a major recessive gene and a number of modifiers. Progenies of crosses between resistant F5 populations and commercial cultivars are now available. Molecular markers for race non-specific resistance based on a pea retrotransposon marker system were developed. It is suggested that the combination of race specific and race non-specific resistance provides the optimal genetic background for the maximum expression of resistance to all races of the pathogen in all plant parts and under field conditions. The second measure was biological control by endophytic bacteria. Studies on the ecology of endophytic bacteria in pea and identification of efficient indigenous colonizers for potential application in biocontrol have been made. Endophytic population levels were in the range 10 3 -10 6 CFU/g fresh tissue in roots and stems. There was a predominance of Gram-negative bacteria, particularly Pseudomonas sp. and Pantoea agglomerans. Arthrobacter sp. and Curtobacterium sp. were the main Gram-positive bacteria. Factors such as soil type, plant genotype and crop growth stage may significantly influence the diversity and population levels of endophytic bacteria. Future research should focus on the combination and testing of elite breeding lines with selections of disease suppressive endophytic isolates under a variety of field conditions in order to obtain an efficient and durable performance in commercial agriculture.</p

Herbaceous plants in the understory of a pitch canker-affected Pinus radiata plantation are endophytically infected with Fusarium circinatum

[EN] Fusarium circinatum was recently detected as an endophyte in grasses causing no apparent damage. Our goal was to describe the endophytic colonization of herbaceous host plants growing in a plantation of Pinus radiata with symptoms of pitch canker disease, which may act as a reservoir of inoculum. We detected the fungus in five species of dicot families (Asteraceae, Lamiaceae, Rosaceae), in addition to two species in the Poaceae. The fungus was found in the aerial part of non-symptomatic hosts, so we describe E circinatum as an endophyte that is mainly transmitted by spores through the air. It was also detected in Hypochaeris radicata seeds, suggesting the potential occurrence of vertical transmission. An analysis of microsatellite markers showed a unique haplotype regardless of whether the isolates' origin was pine cankers or non-symptomatic herbaceous plants. Thus, the same genotype can adopt a pathogenic or endophytic lifestyle. We conclude that non -symptomatic plants can act as reservoirs of inoculum: pine seedlings can be infeded from senescent tissue of non-symptomatic hosts colonized by the fungus. (C) 2017 Elsevier Ltd and British Mycological Society. All rights reserved.We acknowledge Maite Morales Clemente for her excellent technical assistance and Inigo Zabalgogeazcoa for his helpful suggestions. Laura Hernandez was supported by a fellowship from INIA (FPI-INIA). Financial support for this research was provided by projects RTA2012-00015-C02-01 and RTA2013-00048-C03-01 (Programa Estatal I + D + i, INIA, Spain).Hernandez-Escribano, L.; Iturritxa, E.; Elvira-Recuenco, M.; Berbegal Martinez, M.; Campos, J.; Renobales, G.; García, I.... (2018). Herbaceous plants in the understory of a pitch canker-affected Pinus radiata plantation are endophytically infected with Fusarium circinatum. Fungal Ecology. 32:65-71. https://doi.org/10.1016/j.funeco.2017.12.001S65713

The increasing threat to European forests from the invasive foliar pine pathogen, Lecanosticta acicola

European forests are threatened by increasing numbers of invasive pests and pathogens. Over the past century, Lecanosticta acicola, a foliar pathogen predominantly of Pinus spp., has expanded its range globally, and is increasing in impact. Lecanosticta acicola causes brown spot needle blight, resulting in premature defoliation, reduced growth, and mortality in some hosts. Originating from southern regions of North American, it devastated forests in the USA's southern states in the early twentieth century, and in 1942 was discovered in Spain.Derived from Euphresco project 'Brownspotrisk,' this study aimed to establish the current distribution of Lecanosticta species, and assess the risks of L. acicola to European forests. Pathogen reports from the literature, and new/ unpublished survey data were combined into an open-access geo-database (http://www.portaloff orestpathology.com), and used to visualise the pathogen's range, infer its climatic tolerance, and update its host range. Lecanosticta species have now been recorded in 44 countries, mostly in the northern hemisphere. The type species, L. acicola, has increased its range in recent years, and is present in 24 out of the 26 European countries where data were available. Other species of Lecanosticta are largely restricted to Mexico and Central America, and recently Colombia.The geo-database records demonstrate that L. acicola tolerates a wide range of climates across the northern hemisphere, and indicate its potential to colonise Pinus spp. forests across large swathes of the Europe. Pre-liminary analyses suggest L. acicola could affect 62% of global Pinus species area by the end of this century, under climate change predictions.Although its host range appears slightly narrower than the similar Dothistroma species, Lecanosticta species were recorded on 70 host taxa, mostly Pinus spp., but including, Cedrus and Picea spp. Twenty-three, including species of critical ecological, environmental and economic significance in Europe, are highly susceptible to L. acicola, suffering heavy defoliation and sometimes mortality. Variation in apparent susceptibility between reports could reflect variation between regions in the hosts' genetic make-up, but could also reflect the signif-icant variation in L. acicola populations and lineages found across Europe. This study served to highlight sig-nificant gaps in our understanding of the pathogen's behaviour.Lecanosticta acicola has recently been downgraded from an A1 quarantine pest to a regulated non quarantine pathogen, and is now widely distributed across Europe. With a need to consider disease management, this study also explored global BSNB strategies, and used Case Studies to summarise the tactics employed to date in Europe

Effect of Acinetobacter sp on Metalaxyl Degradation and Metabolite Profile of Potato Seedlings (Solanum tuberosum L.) Alpha Variety

One of the most serious diseases in potato cultivars is caused by the pathogen Phytophthora infestans, which affects leaves, stems and tubers. Metalaxyl is a fungicide that protects potato plants from Phytophthora infestans. In Mexico, farmers apply metalaxyl 35 times during the cycle of potato production and the last application is typically 15 days before harvest. There are no records related to the presence of metalaxyl in potato tubers in Mexico. In the present study, we evaluated the effect of Acinetobacter sp on metalaxyl degradation in potato seedlings. The effect of bacteria and metalaxyl on the growth of potato seedlings was also evaluated. A metabolite profile analysis was conducted to determine potential molecular biomarkers produced by potato seedlings in the presence of Acinetobacter sp and metalaxyl. Metalaxyl did not affect the growth of potato seedlings. However, Acinetobacter sp strongly affected the growth of inoculated seedlings, as confirmed by plant length and plant fresh weights which were lower in inoculated potato seedlings (40% and 27%, respectively) compared to the controls. Acinetobacter sp also affected root formation. Inoculated potato seedlings showed a decrease in root formation compared to the controls. LC-MS/MS analysis of metalaxyl residues in potato seedlings suggests that Acinetobacter sp did not degrade metalaxyl. GC–TOF–MS platform was used in metabolic profiling studies. Statistical data analysis and metabolic pathway analysis allowed suggesting the alteration of metabolic pathways by both Acinetobacter sp infection and metalaxyl treatment. Several hundred metabolites were detected, 137 metabolites were identified and 15 metabolic markers were suggested based on statistical change significance found with PLS-DA analysis. These results are important for better understanding the interactions of putative endophytic bacteria and pesticides on plants and their possible effects on plant metabolism

Efficiency of procedures for induction and cultivation of Pseudomonas syringae pv. pisi L-form

The L-form of Pseudomonas syringae pv. phaseolicola has been proved to induce resistance to bean halo blight.Various procedures were tested to induce the L-form of Pseudomonas syringae pv. pisi for its potential use as biocontrol agent of pea bacterial blight. Cell-wall deficient cells were induced in a liquid medium with penicillin following a protocol described for P. s. pv. phaseolicola. Cell growth on solid induction medium developed as typical granular and vacuolated structures, and characteristic colonies were observed in the first transfer. However, there was poor growth in subsequent transfers and some reversion to the parental type. To improve the induction procedure, the following new procedures were applied: (1) viability of cells was monitored during induction. The optimum induction time in liquid medium with penicillin was lower for pv. pisi than for pv. phaseolicola. Viability of L-forms in solid induction medium with penicillin was low and decreased in time. (2) the inducer ticarcillin was combined with clavulanic acid, which prevented the reversion to the parental type and (3) a range of concentrations of penicillin and ticarcillin/clavulanic acid was applied by the spiral gradient endpoint method for calculation of minimum inhibitory concentrations (MIC). Based on the results from these tests an induction method for P. s. pv. pisi L-form is proposed and the relevance of L-form is discussed for practice

Efficiency of procedures for induction and cultivation of Pseudomonas syringae pv. pisi L-form

The L-form of Pseudomonas syringae pv. phaseolicola has been proved to induce resistance to bean halo blight.Various procedures were tested to induce the L-form of Pseudomonas syringae pv. pisi for its potential use as biocontrol agent of pea bacterial blight. Cell-wall deficient cells were induced in a liquid medium with penicillin following a protocol described for P. s. pv. phaseolicola. Cell growth on solid induction medium developed as typical granular and vacuolated structures, and characteristic colonies were observed in the first transfer. However, there was poor growth in subsequent transfers and some reversion to the parental type. To improve the induction procedure, the following new procedures were applied: (1) viability of cells was monitored during induction. The optimum induction time in liquid medium with penicillin was lower for pv. pisi than for pv. phaseolicola. Viability of L-forms in solid induction medium with penicillin was low and decreased in time. (2) the inducer ticarcillin was combined with clavulanic acid, which prevented the reversion to the parental type and (3) a range of concentrations of penicillin and ticarcillin/clavulanic acid was applied by the spiral gradient endpoint method for calculation of minimum inhibitory concentrations (MIC). Based on the results from these tests an induction method for P. s. pv. pisi L-form is proposed and the relevance of L-form is discussed for practice