First report of Agrobacterium tumefaciens causing crown gall disease of roselle (Hibiscus sabdariffa L.) in Taiwan

Roselle (Hibiscus sabdariffa L.) plants, whose calyces are used for production of beverages or jams, are mainly cultivated in Taitung County of eastern Taiwan. Since 2016, large crown galls were observed on the roselle plants in the commercial plantations at Taimali and Jinfong Townships of Taitung County. A follow-up survey in July and August of 2017 revealed spreading of this disease to the neighboring areas including Beinan and Dawu Townships. Disease incidence was estimated to be 0.6-10%. Galls of varying sizes (2-15 cm in diameter) were usually found on the roots and crowns of the roselle plants, starting with small swellings at the infection sites. Galls were light-colored, and smooth and tender in texture at the early stage, but later turned dark-colored, and appeared rough and woody. In some cases, adventitious roots extruding from the larger crown galls could be seen. Isolation of the causal agent was performed by quadrantally streaking bacterial suspension made from surface-sterilized, macerated galls on trypticase soy agar (TSA). After incubating at 28°C for 5 days, single colonies were transferred onto new TSA plates for further cultivation at 28°C. Finally, circular, convex, viscous and milky white colonies with smooth surface similar to colony morphology of Agrobacterium tumefaciens C58 were obtained for further identification. First, all six candidate isolates (TZ-1, TL1-2, TL2-1, TD1-1, TD1-24 and TD2-1) were identified as Agrobacterium spp. using the partial sequences of the 16S rRNA gene (accession numbers MW205820 to MW205825 in the GenBank database). The selected isolates also showed some biochemical and physiological characteristics similar to A. tumefaciens, including oxidase positive, growth at 35°C and in 2% NaCl, and alkalinity from litmus milk. Moreover, they were tested negative for utilization of citrate and acid production on potato dextrose agar (PDA) supplemented with calcium carbonate. Under a transmission electron microscope, the bacterium was rod-shaped and possessed peritrichous flagella. By means of multiplex PCR using primers designed for differentiation of Agrobacterium rubi, Agrobacterium vitis and Agrobacterium biovars 1 and 2, a 184 bp product was detected in all six isolates, indicating that they all belong to Agrobacterium biovar 1. Furthermore, the recA allele of each isolate was PCR amplified using primers F2898/F2899, and recA sequence analysis assigned all six isolates to A. tumefaciens genomospecies G7 (GenBank accession numbers MZ570905-MZ570910). Pathogenicity assay was carried out by inoculating the stems of 2-week-old roselle seedlings through wounds made with a sterile needle with bacteria on it. The inoculated seedlings were kept in plastic bags to maintain high humidity. Symptoms similar to those observed in the field developed at the inoculation sites after 7 days, and Koch’s postulates were fulfilled when the bacteria re-isolated from the galls were also identified as A. tumefaciens genomospecies G7 using recA gene sequence analysis. To our knowledge, this is the first report of crown gall disease caused by A. tumefaciens on Hibiscus sabdariffa in Taiwan. This disease may potentially damage the roselle industry if no action is taken to stop its spreading. Identification of the causal agent of roselle crown gall disease could help us further investigate its ecology and develop integrated pest management strategies for prevention of this disease in the future.

Revisiting the taxonomy of Allorhizobium vitis (i.e. Agrobacterium vitis) using genomics - emended description of All. vitis sensu stricto and description of Allorhizobium ampelinum sp. nov

Allorhizobium vitis (formerly named Agrobacterium vitis or Agrobacterium biovar 3) is the primary causative agent of crown gall disease of grapevine worldwide. Whole-genome sequence comparisons and phylogenomic analysis of various All. vitis strains clearly indicated that All. vitis is not a single species, but represents a species complex composed of at least four genomic species. Thus, we amended the description of All. vitis which now refers to a restricted group of strains within the All. vitis complex (i.e. All. vitis sensu stricto) and proposed a description of a novel species All. ampelinum sp. nov. The type strain of All. vitis sensu stricto remains the existing type strain of All. vitis, K309T (= NCPPB 3554T =HAMBI 1817T = ATCC 49767T = CIP 105853T = ICMP 10752T = IFO 15140T = JCM 21033T = LMG 8750T = NBRC 15140T). The type strain of All. ampelinum sp. nov. is S4T (= DSM 112012T = ATCCBAA-846T). This genome-based classification was supported by differentiation of strains based on a MALDI-TOF MS analysis. We also identified gene clusters specific for All. vitis species complex, All. vitis sensu stricto and All. ampelinum, and attempted to predict their function and their role in ecological diversification of these clades, some of which were experimentally validated. Functions of All. vitis species complex-specific genes convergently indicate a role in adaptation to different stresses, including exposure to aromatic compounds. Similarly, All vitis sensu stricto-specific genes also confer the ability to degrade 4-hydroxyphenylacetate and a putative compound related to gentisic acid, while All. ampelinum-specific genes have putative functions related to polyamine metabolism and nickel assimilation. This suggests that these species have differentiated ecologies, each relying on specialized nutrient consumption or toxic compound degradation to adapt to their respective niche. Moreover, our genome-based analysis indicated that Allorhizobium and the “R. aggregatum complex” represent separate genera of the family Rhizobiaceae.

Bazı Üzüm Çeşit ve Amerikan Asma Anaçlarında Sıcak Su Uygulamasının Aşılı Çeliklerde Kallus Oluşumu Üzerine Etkileri

Bu çalışmada, sağlıklı asma fidanı üretiminde Agrobacterium vitis eleminasyonu amacıyla yapılan sıcak su uygulamasının aşılı çeliklerde kallus oluşumuna etkisinin belirlenmesi amaçlanmıştır. Çalışmada 99R ve Ramsey anaçlarına ait aşılık çelikler ile Sultani Çekirdeksiz ve Yalova İncisi çeşitlerine ait kalemler materyal olarak kullanılmıştır. Aşılık çelik ve kalemler 50 oC de 15, 30 ve 45 dakikalık sıcak su uygulamaların ardından masabaşı omega aşılama yöntemi ile aşılanmıştır. Ramsey anacına Sultani Çekirdeksiz çeşidi, 99R anacına ise Sultani Çekirdeksiz ve Yalova İncisi çeşitleri aşılanmıştır. Bunun ardından aşılı çelikler standart aşılı fidan üretim programına alınarak kallus oluşumu durumu değerlendirilmiştir. Sıcak su uygulaması yapıldıktan sonra 21 gün gelişmeye bırakılmış aşılı çeliklerde kallus oluşumları değerlendirilmiştir. Kombinasyonlar arasında önemli farklar olduğu belirlenmiştir. Sıcak su uygulamasında kallus oluşturma durumu uygulama süresinden çok anaç ve çeşide bağlı görünmektedir.

A novel culture-independent method to search unknown dominant bacterial groups and its application to microbial analysis of membrane bioreactors

ABSTRACTAs we could not get numerical information for unknown unculturable microorganisms through conventional culture-independent analysis methods such as next-generation sequencing, or real time PCR, we developed an original culture-independent method, and searched the numerically dominant bacteria in three industrial membrane bioreactors for livestock farms.Although Actinobacteria was the numerically dominant phylum (9.3×105MPN/mL) on 6/August/2014 in the MBR of A farm, when a bacteria with the same genotype to Arthrobacter sp. (AF197047; 4.3×105MPN/mL), and those similar to Burkholderia sp. (AB299593; 4.3×105MPN/mL) were the numerically dominant, after about 13 months (24/October/2015) a number of the Arthrobacter genotype increased to 930×105 MPN (230 times) and become dominant, and those similar to the Microbacterium sp. (AM403628) increased to 92×105MPN, while that of the Burkholderia genotype disappeared. In the other MBR of B farm, bacteria having a similar genotype to Enshifer sp. (AB195268, CP000738), or Shinorhizobium sp. (AF227755, AB195268), or Mesorhizobium sp. (BA000012, Mso.tians29), or Agrobacterium vitis (D12795) was dominant on 18/August/2015 (24×105 MPN) and 30/August/2015 (15.5×105 MPN). In the other MBR of C farm (9/October/2015), bacteria having a similar genotype to uncultured Betaproteobacteria (AY921864) was dominant (430×105MPN), followed by uncultured bacterium (74×105MPN ; AM268745), and Mycobacteriaceae (AB298730), or Propionibacteriaceae (AB298731) (7.4 ×105MPN). There was no common bacterial groups among tested three MBRs. Present results indicated that different kinds of homogeneous bacteria were numerically dominant in the three tested membrane bioreactors, where their numbers and ratios were varied with the duration of the driving periods.IMPORTANCEAlthough the conventional molecular-based culture independent methods have been used in place of traditional culture-based methods for microbiological research and expanded information of unculturable low-abundance bacterial groups, not all of them were always highly active in the environment and it was difficult to search for microorganisms among them which were highly active and play an important role in the environment. As numerical data of each bacteria might become an important index to know their activity in environment, we had created a novel culture-independent enumeration method for numerically dominant unidentified bacteria. Through the method, we found that different kinds of homogeneous bacteria were numerically dominant in the three tested membrane bioreactors, whose numbers were high enough to affect the performance of the reactor as a single strain. The method was found useful to specify and trace unknown numerically dominant bacterial groups in a culture independent manner.

Two Novel Genomospecies in the Agrobacterium tumefaciens Species Complex Associated with Rose Crown Gall

In this study, we explored the pathogenicity and phylogenetic position of Agrobacterium spp. strains isolated from crown gall tissues on annual, perennial, and ornamental plants in Iran. Of the 43 strains studied, 10 strains were identified as Allorhizobium vitis (formerly Agrobacterium vitis) using the species-specific primer pair PGF/PGR. Thirty-three remaining strains were studied using multilocus sequence analysis of four housekeeping genes (i.e., atpD, gyrB, recA, and rpoB), from which seven strains were identified as A. larrymoorei and one strain was identified as A. rubi (Rer); the remaining 25 strains were scattered within the A. tumefaciens species complex. Two strains were identified as genomospecies 1 (G1), seven strains were identified as A. radiobacter (G4), seven strains were identified as A. deltaense (G7), two strains were identified as A. nepotum (G14), and one strain was identified as “A. viscosum” (G15). The strains Rnr, Rnw, and Rew as well as the two strains OT33 and R13 all isolated from rose and the strain Ap1 isolated from apple were clustered in three atypical clades within the A. tumefaciens species complex. All but eight strains (i.e., Nec10, Ph38, Ph49, fic9, Fic72, R13, OT33, and Ap1) were pathogenic on tomato and sunflower seedlings in greenhouse conditions, whereas all but three strains (i.e., fic9, Fic72, and OT33) showed tumorigenicity on carrot root discs. The phylogenetic analysis and nucleotide diversity statistics suggested the existence of two novel genomospecies within the A. tumefaciens species complex, which we named “G19” and “G20.” Hence, we propose the strains Rew, Rnw, and Rnr as the members of “G19” and the strains R13 and OT33 as the members of G20, whereas the phylogenetic status of the atypical strain Ap1 remains undetermined.

Analisi di accessioni di vite con marcatori molecolari associati a geni di resistenza a filossera, antracnosi e tumore batterico

Il Centro di Sperimentazione Laimburg nel 2012 ha avviato un progetto denominato RebSelect, in collaborazione con la Provincia Autonoma di Bolzano e con la piattaforma privata Innovitis di Marlengo. Tale progetto ha lo scopo di caratterizzare una collezione che consiste di ca. 150 accessioni di vite di particolare interesse per la presenza di geni di resistenza noti contro malattie fungine e non. Il progetto ha come obiettivo la realizzazione di una banca genetica di varietà di vite e l’utilizzo di una metodica molecolare (MAS, selezione assistita da marcatori) per l’individuazione di geni di resistenza a varie malattie nella collezione; in seguito, l’identificazione di piante resistenti da utilizzare come genitori di incrocio potrà essere utile per la costituzione di nuove varietà resistenti e di alta qualità. In questo lavoro di tesi ci si è concentrati sulle regioni genomiche deputate alla resistenza per fillossera (Viteus vitifoliae), tumore batterico (Agrobacterium vitis) e antracnosi (Elsinoë ampelina), prendendo in considerazione marcatori di tipo microsatellite o di tipo SCAR già pubblicati in letteratura. Dalle analisi eseguite, viene mostrato che i marcatori adatti per la MAS e utilizzabili per l’identificazione di varietà resistenti, impiegate successivamente per ulteriori incroci ai fini di un miglioramento genetico, sono quelli per Rdv1, cioè i marcatori associati alla resistenza a fillossera. Per la resistenza a tumore batterico, Rcg1, i risultati sono discordanti, rendendo questi marcatori inutilizzabili per la MAS: per questa regione, probabilmente, è necessario un mappaggio più fine della zona deputata e una ricerca di marcatori più vicini al gene o ai geni di resistenza al tumore batterico. Infine, per quanto riguarda la resistenza ad antracnosi, l’unico marcatore che sembra essere associato è da scartare completamente poiché da risultati falsi positivi per tutte le accessioni utilizzate nelle analisi.

PCR FINGERPRINTING OF DIVERSE GENOMES FROM BACTERIAL STRAINS USING UNIVERSAL RICE PRIMER (URP)

Twenty primers of 20 mer referred to universal rice primer (URP) were developed from a repetitive sequence of rice genome. URP-PCR protocol employed stringent PCR with high annealing temperature throughout the thermo-cycling reaction, giving high reproducibility. Under the PCR condition, each single URP primer produced characteristic fingerprints from diverse genomes of bacterial species. The universal application of URP-PCR was demonstrated by applying it to 24 strains from Pectobacterium carotovoum subsp. carotovorum, 41 Agrobacterium vitis strains, 3 Xanthomonas spp. 5 Pseudomonas spp, Rhizobium sp. plant pathogenic bacteria, human and animal pathogenic bacterial strains including 6 Escherichia coli, 4 Salmonella spp., 7 Mycobacterium spp and 3 Blucella abortus strains. In addition, thermophilic bacteria were randomly isolated form high temperature compost and their URP-PCR polymorphisms were characterized with genetic relatedness. PCR approach using URP primers will be useful for studying DNA diversity of diverse prokaryotic genomes, especially at inter- and intra species levels.

Quantification of Agrobacterium vitis from Grapevine Nursery Stock and Vineyard Soil using Droplet Digital PCR

Current detection methodologies for Agrobacterium vitis, causing crown gall of grapevines, are time intensive and lack the ability to quantify pathogen abundance in nursery stock and soil. Information on pathogen abundance is a key component to develop management strategies. The aim of this study was to develop a rapid and sensitive quantification assay for grapevine nursery stock and vineyard soil via droplet digital polymerase chain reaction targeting the virA gene. DNA isolated from roots of dormant grapevines originating from nurseries in Germany, California, and Ontario were tested for virA abundance. Bacterial numbers varied with grapevine origin; plants from California had the highest numbers. In addition, rhizosphere soil from two vineyards in the Okanagan valley in British Columbia was tested over a growing season. Sampling time during the season did not affect virA gene abundance. The older vineyard had higher soil A. vitis populations than the younger vineyard. The assay developed here has potential for use in national clean plant programs to prevent import of infected grapevine nursery stock and to test vineyard soil for abundance of the pathogen before planting.