Bacterial wilt-resistant tomato rootstock suppresses migration of <Emphasis Type="Italic">ralstonia solanacearum</Emphasis> into soil

Ralstonia solanacearum, the causal agent of bacterial wilt of tomato, grows in infected plants and migrates from the roots into the soil. We investigated the effectiveness of bacterial wilt-resistant tomato rootstock in reducing the migration of R. solanacearum from susceptible scions into the soil. Rootstock stems were either 3–5 cm tall (low-grafted, LG) or ≥?10 cm tall (high-grafted, HG). After inoculation of scions of the susceptible cultivar (SC) with R. solanacearum below the first flower, there was no difference in disease progression among LG, HG, and ungrafted SC plants, and plants had wilted by 2 weeks. However, the rate of detection of R. solanacearum in the soil of wilted plants was reduced by grafting. The size of the R. solanacearum population in the soil of fully wilted plants increased in the order of HG?<?LG?<?SC. These results show that grafting onto resistant rootstock strongly suppressed the migration of R. solanacearum into the soil by the time of full wilting, and the effect was stronger with a longer rootstock. Migration of R. solanacearum into soil increased with increasing disease severity in SC, LG and HG. These facts suggest that early uprooting of slightly infected plants could control the spread of the bacteria into the soil.     

<Emphasis Type="Italic">Fusarium oxysporum</Emphasis> and <Emphasis Type="Italic">Fusarium avenaceum</Emphasis> associated with yield-decline of pyrethrum in Australia

Fusarium wilt, one of the destructive diseases of cucumber can be effectively controlled by using biocontrol agents such as Trichoderma harzianum. However, the mechanisms controlling T. harzianum-induced enhanced resistance remain largely unknown in cucumber plants. Here we screened the potent T. harzianum isolate TH58 that could effectively control F. oxysporum (FO). Glasshouse efficacy trials also showed that TH58 decreased disease incidence by 69.7 %. FO induced ROS over accumulation, while TH58 inoculation suppressed ROS over accumulation and improved root cell viability under F. oxysporum infection. TH58 inoculation could reverse the FO-induced cell division block and regulate the proportional distribution of nuclear DNA content through inducing 2C fraction. Moreover, the expression levels of cell cycle-related genes such as CDKA, CDKB, CycA, CycB, CycD3;1 and CycD3;2 in TH58 - pre-inoculated seedlings were up-regulated compared with those infected with FO alone. Taken together, these results suggest that T. harzianum improved plant resistance against Fusarium wilt disease via alterations in nuclear DNA content and cell cycle-related genes expression that might maintain a lower ROS accumulation and higher root cell viability in cucumber seedlings.     

AlgU contributes to the virulence of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">tomato</Emphasis> DC3000 by regulating production of the phytotoxin coronatine

Pseudomonas syringae pv. tomato DC3000 (Pst DC3000), which causes bacterial speck disease of tomato, has been used as a model pathogen to investigate the molecular basis of plant–pathogen interactions. The function of many potential virulence factors encoded in the Pst DC3000 genome and their modes of action are not fully understood. P. syringae is known to produce the exopolysaccharide alginate. Although AlgU, a sigma factor, is known to regulate the expression of genes such as algD related to alginate biosynthesis, the molecular mechanisms of AlgU in the virulence of Pst DC3000 is still unclear. To investigate the function of AlgU and alginate in plant–bacterial pathogen interactions, we generated ΔalgU and ΔalgD mutants. After inoculation with ΔalgU but not ΔalgD, host plants of Pst DC3000 including tomato and Arabidopsis had milder disease symptoms and reduced bacterial populations. Expression profiles of Pst DC3000 genes revealed that AlgU can regulate not only the expression of genes encoding alginate biosynthesis, but also the expression of genes related to type III effectors and the phytotoxin coronatine (COR). We also demonstrated that the ΔalgU mutant showed full virulence in the Arabidopsis fls2 efr1 double mutant, which is compromised in the recognition of PAMPs. Further, the application of COR was able to restore the phenotype of the ΔalgU mutant in the stomatal response. These results suggest that AlgU has an important role in the virulence of Pst DC3000 by regulating COR production.     

Antagonistic effects of biofilm-forming bacterial strains co-inoculated with blood disease pathogenic strain, <Emphasis Type="Italic">Ralstonia syzygii</Emphasis> subspecies <Emphasis Type="Italic">celebesensis</Emphasis> in banana plants

A blood disease pathogenic strain, Ralstonia syzygii subspecies celebesensis was used to study the possible association of biofilm-forming bacteria with the development and severity of blood disease in banana plants. Therefore, the objective of this study was to determine the effects of mono-culture and co-culture inoculation of isolated biofilm-forming bacteria with the blood disease pathogen in banana pseudostems in glasshouse conditions. Putative biofilm-forming bacteria were isolated from an infected banana plant and were further identified using 16SrRNA sequencing. Four isolates, identified as Enterobacter hormaechei, Enterobacter cloacae, Kosakonia radicincitans and Klebsiella pneumoniae, were inoculated as a mono- and co-culture with R. syzygii subsp. celebesensis into 2 months old banana plants. The observation after the 8 weeks of post inoculation showed that plants which were co-inoculated with the pathogen and K. radicincitans, a biofilm-forming bacterium, were the most susceptible towards the infection. In contrast, plants under two treatments (which were co-inoculated with the pathogen and E. cloacae and the pathogen with E. hormaechei) were less susceptible towards the infection. This study revealed the antagonistic effects of two biofilm-forming strains which reduced the severity of infection caused by the pathogenic agent. Scanning electron micrographs of the cross section of plant rhizomes indicated the dissimilarity of adhesion and host colonization conditions of the pathogen in each infected plant from different treatments.     

Visual detection of <Emphasis Type="Italic">Didymella bryoniae</Emphasis> in cucurbit seeds using a loop-mediated isothermal amplification assay

A method was developed using a Loop-mediated isothermal amplification assay (LAMP) for detecting Didymella bryoniae in cucurbit seeds. The LAMP primers were designed based on the DNA-dependent RNA polymerase II RPB140 gene (RPB2) from D. bryoniae. Calcein was used as an indicator for the endpoint visual detection of DNA amplification. The LAMP assay was conducted in isothermal (65 °C) conditions within 1 h. The detection threshold of the LAMP assay was 10 pg of genomic DNA and D. bryoniae was detected in 100 % of artificially infested seedlots with 0.05 % infestation or greater. With the LAMP assay, 16 of 60 watermelon and muskmelon seedlots collected from Xinjang province were determined to be positive for D. bryoniae. In contrast, a real-time PCR assay determined that 11 of the 60 seedlots from Xinjiang province were positive for the pathogen. These results showed that the LAMP technique was simple, rapid and well suited for detecting D. bryoniae DNA, especially in seed health testing.     

Possibilities of biological and chemical control of Verticillium wilt in pepper

In pathogen populations in Serbia, the incidence, pathogenic and morphological characters ofVerticillium spp. were studied. Biological and chemical control ofVerticillium was investigated in pepper plants (Capsicum annuum L. cv. ‘Soroksari’) with the biofungicide Polyversum^® (Pythium oligandrum) and the conventional fungicides benomyl and propamocarb-hydrochloride. On the basis of macroscopic and microscopic characters of the isolates originating from eight localities in Serbia, it was established that they apparently belong to the speciesVerticillum dahliae. The isolates differed in their pathogenic characters. However, all of them caused marked wilting symptoms on pepper plants 40 days after inoculation, conducted when there were more than nine fully developed leaves on the primary stem. The fungicides were applied either before or after inoculation. Benomyl was the most efficient fungicide in wilt control (88.2% when applied after inoculation and 94.6% when applied before inoculation). Polyversum proved more efficient (66.6%) when applied before rather than after inoculation. Propamocarb-hydrochloride provided sufficient Verticillium wilt control; its efficacy and that of Polyversum were similar, and less efficient than benomyl, but still significantly different from the disease control.     

Application of matrix-assisted laser desorption ionization time-of-flight mass spectrometry for rapid and accurate identification of <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> and <Emphasis Type="Italic">Ralstonia pseudosolanacearum</Emphasis>

Ralstonia solanacearum “species complex” (RSSC) represents soil-borne plant pathogenic bacteria, consisting of diverse and widespread strains that cause bacterial wilt on a wide range of host plants. A recent polyphasic taxonomic study has divided the RSSC into three bacterial species; Ralstonia pseudosolanacearum (phylotypes I and III), Ralstonia solanacearum (phylotype II) and Ralstonia syzygii (phylotype IV). Currently, standard identification of RSSC in plant health laboratories mainly relies on performance of two tests that are based on a different principle. However, these tests are inadequate to precisely discriminate among the three bacterial species in the RSSC. The accurate identification of each of the three bacterial species in the RSSC requires additional molecular tests, including a phylotype determination. These methodologies are labor-intensive, time consuming and rather impractical for routine identification purposes in a plant health laboratory. We explored the potential for an accurate identification of R. pseudosolanacearum (phylotypes I and III) and R. solanacearum (phylotype II) in RSSC, upon implementation of the MALDI-TOF MS tool, and after the creation and validation of an in-house database supplementing the commercial database and covering the entire known genetic diversity in RSSC. MALDI-TOF MS is an emerging approach for identification of bacterial plant pathogens and has been shown to be robust and reproducible. Additionally, when compared to the conventional microbial identification methods it is shown to be less laborious and less expensive. Validation data demonstrated that our in-house database (Mass Spectra Profiles, MSPs) was very specific resulting in the rapid and accurate identification of Ralstonia solanacearum (phylotype II), and Ralstonia pseudosolanacearum (phylotypes I and III). Additionally, no false positive results were obtained with our in-house database for other related Ralstonia sp., such as the R. picketii isolate PD 3286, or for the Pseudomonas syringae and Pseudomonas spp. isolates.     

Association of <Emphasis Type="Italic">Pantoea ananatis and Pantoea agglomerans</Emphasis> with leaf spot disease on ornamental plants of Araceae Family

During a survey in 2011–2012, three ornamental plants of Araceae namely Aglaonema nitidum, Syngonium podophyllum and Dieffenbachia amoena showing foliar disease symptoms were collected from central region of Iran. Infected plants exhibited spots on their leaves which appeared as yellow and water-soaked with chlorotic haloes and necrotic center. To investigate the etiology of this disorder, symptomatic leaves were collected from affected plants and six bacterial strains (B2Y, J3Y, SY, E60Y, E68Y and E5MM) were isolated and identified as Pantoea ananatis or P. agglomerans based on morphological, physiological, biochemical and molecular characters. The pathogenicity tests of the isolates demonstrated that they were not host specific. Furthermore, 16S rRNA gene sequencing revealed that the strains were phylogenetically closely related to genus Pantoea. Multilocus sequence analysis (MLSA) of concatenated partial atpD, gyrB and rpoB gene sequences of the six isolates showed a high similarity of B2Y, J3Y, and SY strains to P. ananatis and also of E60Y, E5MM and E68Y strains to P. agglomerans. These results were confirmed by phylogenetic analysis. To the best of our knowledge, this is the first report of leaf spot and necrosis of A. nitidum, S. podophyllum and D. amoena caused by the genus Pantoea.     

Efficacy of some rhizospheric and endophytic bacteria <Emphasis Type="Italic">in vitro</Emphasis> and as seed coating for the control of <Emphasis Type="Italic">Fusarium culmorum</Emphasis> infecting durum wheat in Tunisia

Sixty two rhizospheric and endophytic bacterial strains were evaluated for their biocontrol effect on two aggressive Fusarium culmorum isolates (Fc2 and Fc3). We observed that 35 % and 23 % of the tested strains inhibited the in vitro growth of Fc2 and Fc3 respectively. The observed antagonism was due to inhibition by contact (13–19 % of the strains) or at distance (10–16 % of the strains) for both fungal isolates. Some of the antagonistic bacteria showed the ability to produce diffuse and/or volatile compounds that inhibit the growth, the sporulation and macroconidia germination of F. culmorum. None of the tested antagonistic bacteria showed chitinase activity on synthetic medium. The sequencing of the 16S rDNA genes of some antagonistic bacteria showed that they belong to the genera Bacillus, Pseudomonas and Microbacterium. The double inoculation of durum wheat seeds by the antagonistic bacterial strains (B13, B18, BSE1, BSE3 and B16E) and the two F. culmorum isolates showed that germination and seedling vigor were generally improved in vitro. The percentage of infected seeds was also reduced. In greenhouse trials, the biocontrol effectiveness of F. culmorum was dependant from the virulence of the fungal strain and the specificity of the antagonistic interaction between bacterial and fungal strains. The bacterial strains B18 and B16E reduced F. culmorum infection on durum wheat plants probably due to their antagonistic and plant growth promoting activities and they may be used in a mixture as seed biopriming inoculum for plant growth bio-promoting and Fusarium wheat diseases biocontrol.     

A chalcone synthase controls the verticillium disease resistance response in both <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and cotton

Verticillium wilt is a devastating disease caused by the soil-borne fungus Verticillium dahliae that causes severe wilt symptoms in more than 400 plant species, including economically important cotton. However, the molecular mechanism of plant resistance to Verticillium remains unclear. In this study, we identified an Arabidopsis mutant, vsad1 (verticillium sensitive and anthocyanin deficient 1), which showed more serious disease symptoms such as discoloration and chlorosis than wild-type Arabidopsis. vsad1 is a previously identified allele of the transparent testa 4 gene (tt4), which encodes chalcone synthase (CHS), a key enzyme involved in the biosynthesis of flavonoids. Our results showed that VSAD1 expression was induced in response to Verticillium dahliae infection. Overexpression of VSAD1 partially recovered the anthocyanin accumulation phenotype of the vsad1–1 mutant. The concentration of V. dahliae increased and ROS accumulation decreased in the vsad1 mutant after infection with V. dahliae. Knockdown of the homologous gene GhCHS in cotton plants increased their susceptibility to V. dahliae infection. Thus, we conclude that VSAD1 is involved in the regulation of plant resistance to Verticillium wilt.     

Chitinolytic <Emphasis Type="Italic">Streptomyces griseorubens</Emphasis> E44G enhances the biocontrol efficacy against <Emphasis Type="Italic">Fusarium</Emphasis> wilt disease of tomato

Streptomyces griseorubens E44G is a chitinolytic bacterium isolated from cultivated soil in Saudi Arabia (a hot, arid climatic region). In vitro, antifungal potential of S. griseorubens E44G was assessed against the phytopathogenic fungus, Fusarium oxysporum f. sp. lycopersici (the causative agent of the Fusarium wilt disease of tomato). An inhibition zone of 24 mm was recorded. The chitinolytic activity of S. griseorubens E44G was proved when the colloidal chitin agar plate method was used. A thermostable chitinase enzyme of 45 kDa molecular weight was purified using gel filtration chromatography. The optimum activity was obtained at 60 °C and pH 5.5. The purified enzyme has shown a very pronounced activity against the phytopathogenic fungus, F. oxysporum. The molecular characterization of the chitinase gene indicated that it consists of 1218 bp encoding 407 amino acids. The phylogentic analysis based on the nucleotide DNA sequence and the deduced amino acids sequence showed high similarity percentages with other chitinases isolated from different Streptomyces species. In the field evaluation, application of both S. griseorubens E44G treatments significantly increased all tested growth and yield parameters and decreased the disease severity compared with the infected-untreated tomato plants suggesting potential as a biocontrol agent.     

Distribution and persistence of <Emphasis Type="Italic">Verticillium dahliae</Emphasis> in the xylem of Norway maple and European ash trees

Verticillium dahliae colonizes the xylem vessels of susceptible host plants. Hence it can be expected that the distribution of the fungus as well as disease progress will be influenced by the anatomy of the xylem of that host. Here, we studied the spatial and temporal distribution of V. dahliae in relation to recovery from disease symptoms in young European ash (Fraxinus excelsior) and Norway maple (Acer platanoides) trees that differ in vascular anatomy. Quantifying the amount of V. dahliae DNA at different heights in the stem of inoculated trees at different time points after inoculation showed that, in the year of inoculation, the speed of colonization of these two species by V. dahliae was similar. Nevertheless, in the year after inoculation disease incidence and also quantities of V. dahliae detected in maple trees were significantly higher than in ash trees, suggesting that the xylem of ash trees is much less supportive for growth and survival of V. dahliae than that of maple trees. Moreover, in this second year V. dahliae could not be re-isolated from the wood of ash trees that had recovered from disease and was only rarely detected by PCR, only in xylem of the previous year and never in the current xylem. In contrast, V. dahliae easily was detected in the wood of diseased ash and maple trees. Furthermore, despite the presence of a layer of parenchyma cells between growth rings in ash trees, in symptomatic ash trees V. dahliae was present in the xylem of the new growth ring. We also observed that V. dahliae can move downward from the point of inoculation into the root collar, which possibly provides a way for infection of new growth rings by circumventing the physical barriers within the stem xylem.     

A sensitive biosensor based on gold nanoparticles to detect <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> in soil

Ralstonia solanacearum, the devastating causal agent of potato bacterial wilt, is a soil-borne bacterium that can survive in the soil for a long time. The development of sensitive on-field detection methods for this pathogen is highly desirable due to its widespread host range and distribution. A novel nanobiosensor was thus developed to detect unamplified genomic DNA of R. solanacearum in farm soil. Gold nanoparticles functionalized with single-stranded oligonucleotides served as a probe to detect R. solanacearum genomic DNA. The advantages of this strategy include rapidity, facile usage and being a visual colorimetric method.     

Management of laurel wilt of avocado,caused by <Emphasis Type="Italic">Raffaelea lauricola</Emphasis>

Laurel wilt is caused by Raffaelea lauricola, a nutritional symbiont of an Asian ambrosia beetle, Xyleborus glabratus. American members of the Lauraceae plant family are most susceptible and 300 million trees have been killed by the disease in the southeastern USA since 2003. Recently, commercial production of an important crop in the laurel family, avocado (Persea americana), has been affected in southern Florida. We summarize studies in which diverse measures were tested for managing the disease. In all studies, trees were treated with potential laurel wilt control measures and subsequently inoculated with R. lauricola. On potted plants in greenhouse experiments, commercial nutritional products (Greenstim and Keyplex 350) and SAR products (Agri-Fos and Nutri-Phite), when applied as soil drenches or foliar sprays, had either no impact on, or increased laurel wilt symptom development compared to non-treated control treatments. Bark applications of Tilt (a propiconazole fungicide for which emergency registration had been obtained in 2010) in a surfactant (Pentrabark) enabled significant laurel wilt protection in greenhouse studies on small potted plants, but Pentrabark and other surfactants moved little propiconazole into the xylem of fruit-bearing trees in field studies. In efficacy studies in the field, Propiconazole Pro (an injectable formulation of propiconazole), Tilt, and two experimental formulations of another triazole fungicide, tebuconazole, decreased the development of laurel wilt compared to nontreated control trees when applied as undiluted injections into branches and scaffold limbs (microinjection), or injection of dilute fungicide into tree flare roots (macroinfusion). However, symptoms developed in all treated trees by 10–11 months after inoculation with R. lauricola, indicating that trees would need to be re-treated at least on an annual basis. Regardless of how fungicides were applied, insignificant levels of different active ingredients entered fruit. Although fungicide treatment of fruit-bearing avocado trees is not a concern for food safety and several triazole and DMI fungicides can protect avocado trees from laurel wilt, cost-effective measures with which the xylem could be loaded with and protected by these products remains a challenge. Management of laurel wilt in commercial avocado production areas is discussed.     

Histological observation of cucumber infected with <Emphasis Type="Italic">Corynespora cassiicola</Emphasis>

Erwinia amylovora is the causative agent of fire blight, which is a destructive bacterial disease of rosaceous plants. In Hungary Erwinia amylovora (Burrill) Winslow et al. was first detected in 1996. Since the appearance of fire blight, E. amylovora samples have been collected from different host plats from various geographic locations. A motif of eight nucleotides (ATTACAGA) is repeated 3–15 times in the PstI fragment of the pEa29 plasmid in Erwinia amylovora strains, and represents a valuable tool for strain classification. The number of short-sequence DNA repeats in plasmid pEa29 of 30 Hungarian isolates were determined by PCR assays and they ranged from five to ten. The SSR test is suitable for distinguishing the individual strains between the E. amylovora isolates. The examined isolates showed high pathogenicity on immature pear fruits. Several biochemical techniques, such as miniaturized API 20E, were applied on the samples. Differences were also revealed in microbiological assays like levan formation and colony morphology on semi-selective media. Examining the Hungarian Erwinia amylovora population by molecular analysis we can draw the conclusion that the population consists of different strains, which shows great diversity. E. amylovora is a widespread pathogen in Hungary, which is supported by the 30 strains isolated from various host plants from many parts of the country. The phenotypic diversity-evaluation of the E. amylovora strains showed, that they differ metabolically, like other plant pathogenic bacteria as reported by several authors. This is the first report on the diversity of E. amylovora strains isolated from Hungary.     

The sequevar distribution of <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> in tobacco-growing zones of China is structured by elevation

Bacterial wilt, caused by Ralstonia solanacearum, is a devastating disease resulting in tremendous losses of economic crops such as plants in the Solanaceae. Recent studies showed that R. solanacearum is spreading from the lowlands to the highlands in China. We studied 97 Chinese R. solanacearum strains that were isolated from four tobacco-growing zones over a wide range of elevations using phylotype specific multiplex polymerase chain reaction (Pmx-PCR) and phylogenetic relationships (egl and mutS). The results showed that all isolates belonged to phylotype I, which were further clustered into eight egl-sequence type groups (egl-group, sequevar): sequevars 13, 14, 15, 17, 34, 44, 54, and 55. In addition, Sequevar 55, found from the highlands, was a new/unknown one. Southeast China (Z3) had the largest number of egl-groups, containing six sequevars. The basin of the Yangzi River (Z1) and southwestern China (Z2) contained five egl-groups. The basin of the Huai River (Z4), near the north of China, where slight bacterial wilt occurred recently, contained a single group, sequevar 15. The distribution of sequevars was associated with elevation. Sequevar 15 was over-represented in lowland elevations, while sequevar 54 and the new/unknown one were only found in areas of moderate to high elevations. This finding suggested that the phylotype I strains infecting tobacco were diverse in China and regional integrated control strategies should be considered.     

Panama disease of banana occurred in Miyakojima Island,Okinawa, Japan

In 2016, in Miyakojima, Okinawa, Japan, banana plants (Musa?×?paradisiaca) ‘Shima-banana’ developed yellowing and wilt associated with vascular discoloration of the pseudostems. Fusarium oxysporum, identified based on morphological characters, was frequently isolated from the vascular tissue of the infected plant and reproduced the original symptoms on ‘Shima-banana’ after drench inoculation with a spore suspension. Thereby, we determined that the disease is Panama disease caused by F. oxysporum. This is the first official report of Panama disease (Panama-byo in Japanese) of banana in Japan.     

Description and molecular phylogeny of <Emphasis Type="Italic">Ditylenchus gilanicus</Emphasis> n. sp. (Nematoda: Anguinidae) from northern forests of Iran

Commercial areas containing Eucalyptus plantations have expanded in recent years due to increased demands for pulp, paper and bioenergy. One of the threats that can reduce Eucalyptus production is the eucalyptus rust disease caused by Austropuccinia psidii, a biotrophic fungus that affects a broad range of Myrtaceae. An accurate diagnosis tool for the early detection of rust disease could be useful in breeding programs for selection of resistant plants against rust, in phytosanitary purposes or in rust epidemics studies. The aim of the present work was to develop a SYBR Green-based quantitative real-time PCR (qPCR) assay for the early detection and quantification of A. psidii in Eucalyptus grandis leaves. Three sets of primers based on the A. psidii ribosomal DNA intergenic space region (IGS), beta-tubulin and elongation factor genes were designed and evaluated. The assays using the IGS primer set resulted in the highest detection efficiency, detecting a lower limit of 0.5 pg of A. psidii DNA. Under artificial inoculation in plants, A. psidii was detected immediately after pathogen inoculation until 240 h post-inoculation using qPCR. In field validation of the method, A. psidii was detected using qPCR in naturally infected leaves with or without rust symptoms. This easy and fast method can be used for an efficient detection of A. psidii in E. grandis leaves. The implications of this tool for rust studies are discussed below.