Molecular characterization of Ralstonia solanacearum strains from Ethiopia and tracing potential source of bacterial wilt disease outbreak in seed potatoes

Bacterial wilt, caused by Ralstonia solanacearum, is emerging as a major threat to potato production in Ethiopia, reaching epidemic proportions in the Chencha district recently, with a prevalence of 97% of potato fields in 2015. The recent disease outbreak in the district coincided with a significant introduction of seed potatoes. This research was therefore initiated to genetically characterize the pathogen so as to trace its source, identify its relationship with outbreaks in the rest of the country, and make intervention recommendations. Ralstonia solanacearum isolates were sampled both from seed and ware potato fields in Chencha and from seed potato fields in production regions suspected of being potential sources of the pathogen. Multiplex PCR and phylogenetic analysis of partial endoglucanase gene sequences identified all of the isolates as phylotype IIB sequevar 1. VNTR sequence analysis distinguished 11 different haplotypes, nine of which were unique to the Chencha district. However, one of the haplotypes was common to all seed potato producer regions of Ethiopia except for the Shashemene area. The unique and diverse VNTR haplotypes of the pathogen in Chencha indicates that it is well established in the district. When a geographical map of the VNTR haplotypes was superimposed with the main cross‐regional seed potato distribution pattern of the country, it became evident that the pathogen was being disseminated via latently infected seed from the Holeta‐Jeldu area in the Central Highlands of Ethiopia. Identification of largely uninfected highland districts and multiplication of high‐grade seed potato exclusively in those districts should be given priority.     

Resistance to Xanthomonas perforans race T4 causing bacterial spot in tomato breeding lines

Tomato (Solanum lycopersicum) is the second most important vegetable crop in the world. Bacterial spot (BS) of tomato, caused by four species of Xanthomonas: X. euvesicatoria, X. vesicatoria, X. perforans and X. gardneri, results in severe loss in yield and quality due to defoliation and formation of lesions on fruits, respectively. Currently management practices do not offer effective control under conditions of high disease pressure. Thus, developing BS resistance is a critical priority for tomato growers in order to minimize crop losses. Sixty‐three advanced tomato breeding lines, heirlooms and wild tomato lines with diverse genetic backgrounds were screened under greenhouse and field conditions for BS resistance using X. perforans race T4, which was found to be a prevalent race in North Carolina. Race T4 isolate 9 was used to inoculate the plants by spraying, and disease severity was measured using the Horsfall–Barratt scale. Tomato lines 74L‐1W(2008), NC2CELBR, 081‐12‐1X‐gsms, NC22L‐1 (2008) and 52LB‐1 showed resistance to BS in the field and/or greenhouse trials. These lines were derived from S. pimpinellifolium L3707. Screening L3707 followed by development of a mapping population and mapping resistance genes might be useful for breeding resistance against BS in future breeding programmes.     

MgO-induced defence against bacterial wilt disease in Arabidopsis thaliana

Bacterial wilt, caused by Ralstonia solanacearum, is a devastating soilborne disease in plants that limits the production of many crops worldwide. Although management of bacterial wilt has so far been unsuccessful, enhancing host resistance to the pathogen may be an effective control strategy. Recently, magnesium oxide (MgO) was found to induce defence responses against R. solanacearum in tomato plants. Here, the mechanisms underlying MgO-induced defence responses against R. solanacearum (MgO-i DARS) were investigated using Arabidopsis thaliana as a host plant. MgO-i DARS was confirmed in A. thaliana mutants deficient in jasmonic acid or ethylene signalling pathways as well as in the wildtype (Col-0) plants. In contrast, no MgO-i DARS was found in A. thaliana mutants deficient in the salicylic acid (SA) production (sid2-2) and signalling pathways (tga1-1 and npr-1). MgO treatment led to significant accumulation of SA in both roots and shoots of Col-0. The SA biosynthesis gene isochorismate synthase 1 (ICS1) was induced in roots and shoots of A. thaliana treated with MgO. An NADPH oxidase gene respiratory burst oxidase homolog D (AtRbohD) was up-regulated in both roots and shoots of Col-0 treated with MgO. No MgO-i DARS was observed in A. thaliana mutants deficient in AtRbohD. These results suggest that SA and RBOHD-mediated ROS are pivotal for MgO-i DARS in A. thaliana.     

Involvement of jasmonic acid signalling in bacterial wilt disease resistance induced by biocontrol agent Pythium oligandrum in tomato

When the biocontrol agent Pythium oligandrum (PO) colonizes the rhizosphere, it suppresses bacterial wilt disease in tomato (Solanum lycopersicum cv. Micro‐Tom) caused by Ralstonia solanacearum, and a homogenate of its mycelia exhibits elicitor activity, inducing an ethylene (ET)‐dependent defence response in Micro‐Tom. Since salicylic acid (SA) and jasmonic acid (JA) play an important role in plant defence responses to pathogens, the involvement of SA‐ and JA‐dependent signal transduction pathways in resistance to R. solanacearum was investigated in tomato roots treated with a mycelial homogenate of PO. Bacterial wilt disease was also suppressed in tomato cv. Moneymaker treated with the PO homogenate. However, the SA‐inducible PR‐1(P6) gene was not up‐regulated in either Micro‐Tom or Moneymaker. SA did not accumulate in homogenate‐treated roots in comparison with distilled water‐treated controls, even 24 h after inoculation. Induced resistance against R. solanacearum was not compromised in SA‐non‐accumulating NahG transgenic plants treated with the PO homogenate. On the other hand, the expression of the JA‐responsive gene for the basic PR‐6 protein was induced in both tomato cultivars treated with the PO homogenate. Furthermore, quantitative disease assays showed that the induced resistance against R. solanacearum was compromized in PO homogenate‐treated jai1‐1 mutant plants defective in JA signalling. These results indicated that the JA‐dependent signalling pathway is required for PO‐induced resistance against R. solanacearum in tomato.     

Resistance against Botrytis cinerea in smooth leaf pruning wounds of tomato does not depend on major disease signalling pathways

In high‐tech, heated tomato glasshouses, stem infections caused by Botrytis cinerea usually end up girdling the stem, resulting in plant death and consequently high economic losses. Such infections originate primarily from wounds created during leaf pruning, a common cultural practice in which it is intended to remove leaves completely, resulting in smooth stem wounds. However, hasty leaf pruning often results in numerous petiole stubs accidentally left behind. In this study analysis of disease incidences clearly proved that pruning leaves flush to the stem resulted in absolute resistance of the stem wounds, whereas petiole stubs displayed a high level of susceptibility to B. cinerea. Postponing inoculation of wounds after pruning indicated that development of nearly complete resistance occurs within 48 h after deleafing. Monitoring of the wound wetness period showed that drying of the wound surface is not the cause of the decreased susceptibility, contrary to what was commonly believed. Tomato mutants deficient in disease signalling showed altered phenotypes for susceptibility to B. cinerea, indicating that defences against this pathogen in petiole stubs depend on ethylene signalling. Additionally, the decreased susceptibility of mutants deficient in the biosynthesis of jasmonates and abscisic acid suggest an antagonistic effect of these signal molecules. On the other hand, resistance of smooth stem wounds could not be altered by disruption of salicylic acid, ethylene, jasmonate or abscisic acid signalling. This indicates that this remarkable absolute resistance to B. cinerea does not depend on the major disease signalling pathways.     

Ralstonia solanacearum virulence in tomato seedlings inoculated by leaf clipping

Ralstonia solanacearum is a phytopathogenic bacterium that colonizes the xylem vessels of host plants leading to a lethal wilt disease. Although several studies have investigated the virulence of R. solanacearum on adult host plants, infection studies of this pathogen on the seedling stages of hosts are less common. In a preliminary observation, inoculation of R. solanacearum F1C1 on 6‐ to 7‐day‐old tomato seedlings by a simple leaf‐clip strategy resulted in a lethal pathogenic condition in seedlings that eventually killed these seedlings within a week post‐inoculation. This prompted testing of the effect of this inoculation technique in seedlings from different cultivars of tomato and similar results were obtained. Colonization and spread of the bacteria throughout the infected seedlings was demonstrated using gus‐tagged R. solanacearum F1C1. The same method of inoculating tomato seedlings was used with R. solanacearum GMI1000 and independent mutants of R. solanacearum GMI1000, deficient in the virulence genes hrpB, hrpG, phcA and gspD. Wildtype R. solanacearum GMI1000 was found to be virulent on tomato seedlings, whereas the mutants were found to be non‐virulent. This leaf‐clip technique, for inoculation of tomato seedlings, has the potential to be a valuable approach, saving time, space, labour and costs.     

Detection of Tomato yellow leaf curl virus in imported tomato fruit in northern Europe

Imported tomato fruits infected with Tomato yellow leaf curl virus (TYLCV) were identified on the market in northern Europe using paper‐based FTA Classic Cards (Whatman), polymerase chain reaction (PCR) and partial DNA sequence analysis. Trade tomatoes originating from southern Europe, Africa and the Middle East were sampled in Estonia and Sweden, and tested for infection with begomoviruses. Out of 100 batches analysed with five fruits sampled in each batch (58 batches from Estonia and 42 from Sweden), 20 batches were positive (16 from Estonia and four from Sweden). Rolling circle amplification (RCA) and full‐length genome sequence analysis of one isolate collected in Estonia and one isolate in Sweden, revealed highest nucleotide sequence identity at 99% to TYLCV‐IL for the Estonian isolate and at 97% to TYLCV‐Mld for the Swedish isolate. In this study, TYLCV was identified for the first time in imported tomato fruits on the market in northern Europe. FTA cards proved to be an effective means to collect, extract and store begomovirus DNA from tomato fruits and the subsequent molecular analysis.     

Induction of transient ethylene and reduction in severity of tomato bacterial wilt by Pythium oligandrum

Pythium oligandrum (PO) is a mycoparasite on a wide range of fungi and suppresses diseases caused by fungal pathogens when colonizing the rhizosphere. PO and its cell wall proteins (CWPs) have elicitor activity that induces defence responses in plants. The potential of a mycelial homogenate of PO to suppress bacterial diseases was studied in roots of tomato ( Lycopersicon esculentum cv. Micro-Tom) inoculated with Ralstonia solanacearum . PO-treated plants showed enhanced resistance to R. solanacearum and reduction in severity of wilt symptoms. As ethylene often acts as one of the signal molecules for induced resistance, its production following treatment of tomato roots with the mycelial homogenate or CWP of PO was measured. The level of ethylene in PO- and CWP-treated plants was transiently elevated six- to 11-fold at 4–8 h after treatment, followed by high expression of three basic ethylene-inducible defence-related genes ( PR-2b , PR-3b and PR-5b ). Analysis of PR-5b gene expression in the leaves of PO- and CWP-treated plants suggested that PR gene expression was induced systemically. The expression of LeERF2 and LeETR4 , which confer an ethylene-dependent signalling pathway, was also significantly accelerated by such treatments. These results indicate that PO has the potential to control bacterial wilt disease and that CWP may play an important role in the induction of resistance to R. solanacearum accompanying the activation of the ethylene-dependent signalling pathway.     

Involvement of a vascular hypersensitive response in quantitative resistance to Ralstonia solanacearum on tomato rootstock cultivar LS‐89

Ralstonia solanacearum causes bacterial wilt disease in Solanaceae spp. Expression of the Phytophthora inhibitor protease 1 (PIP1) gene, which encodes a papain‐like extracellular cysteine protease, is induced in R. solanacearum‐inoculated stem tissues of quantitatively resistant tomato cultivar LS‐89, but not in susceptible cultivar Ponderosa. Phytophthora inhibitor protease 1 is closely related to Rcr3, which is required for the Cf‐2‐mediated hypersensitive response (HR) to the leaf mould fungus Cladosporium fulvum and manifestation of HR cell death. However, up‐regulation of PIP1 in R. solanacearum‐inoculated LS‐89 stems was not accompanied by visible HR cell death. Nevertheless, upon electron microscopic examination of inoculated stem tissues of resistant cultivar LS‐89, several aggregated materials associated with HR cell death were observed in xylem parenchyma and pith cells surrounding xylem vessels. In addition, the accumulation of electron‐dense substances was observed within the xylem vessel lumen of inoculated stems. Moreover, when the leaves of LS‐89 or Ponderosa were infiltrated with 10⁶ cells mL^?1 R. solanacearum, cell death appeared in LS‐89 at 18 and 24 h after infiltration. The proliferation of bacteria in the infiltrated leaf tissues of LS‐89 was suppressed to approximately 10–30% of that in Ponderosa, and expression of the defence‐related gene PR‐2 and HR marker gene hsr203J was induced in the infiltrated tissues. These results indicated that the response of LS‐89 is a true HR, and induction of vascular HR in xylem parenchyma and pith cells surrounding xylem vessels seems to be associated with quantitative resistance of LS‐89 to R. solanacearum.