Detection and identification of causal agents of cucurbit bacterial diseases by classical and molecular methods

Familija Cucurbitaceae broji preko 800 biljnih vrsta, od kojih neke imaju široku primenu u ishrani ljudi, prerađivačkoj i farmaceutskoj industriji. S obzirom da su biljke familije Cucurbitaceae posebno osetljive prema nekoliko vrsta fitopatogenih bakterija, u godinama sa uslovima pogodnim za nastanak infekcije, gubici mogu dosegnuti velike razmere. U periodu od 2013-2016. godine, sa različitih lokaliteta širom Srbije, prikupljeni su simptomatični uzorci biljaka familije Cucurbitaceae. Iz obolelog tkiva, izolovano je ukupno 63 soja fitopatogenih bakterija. Sojevi su proučeni primenom standardnih i savremenih metoda. Svi proučavani sojevi bili su Gram-negativni i katalaza pozitivni. Od 63 testirana soja, 61 soj je prouzrokovao pojavu hipersenzitivne reakcije biljaka duvana. Izuzetak su predstavljala 2 soja izolovana iz ploda lubenice sa simptomima bakteriozne mrljavosti koje prouzrokuje vrsta Acidovorax citrulli. Na osnovu patogenih, morfoloških, biohemijsko-fizioloških i molekularnih odlika, izolovani sojevi svrstani su u 3 roda: Acidovorax, Pseudomonas i Pectobacterium. Nepektolitički, oksidaza pozitivni sojevi, koji se razvijaju pri 41˚C, pripadaju rodu Acidovorax. Sojevi na YDC podlozi obrazuju okrugle, ispupčene kolonije, krem boje, sa tendencijom porasta i promene boje kolonija u tamniju. Primenom prajmera BX-L1/BX-S-R2 specifičnih za vrstu A. citrulli, došlo je do umnožavanja fragmenta veličine 279 bp, čime je potvrđena pripadnost izolovanih sojeva ovoj vrsti. Proučavanjem sekvenci 16S rRNK gena, i njihovim poređenjem sa sekvencama iz NCBI baze, takođe je utvrđen visok stepen sličnosti (100%) sa drugim sojevima A. citrulli. U cilju proučavanja genetičkog diverziteta sojeva, primenjen je rep-PCR test. Iako je ispoljena potpuna homogenost sojeva, u BOX-PCR profilu jednog soja, umnožen je fragment koji nije uočen kod ostalih sojeva. Stoga se zaključuje da izolovani sojevi A. citrulli pripadaju populaciji zajedničkog porekla, koja je kod nas dospela zaraženim semenom. Sojevi su ispoljili su visok stepen virulentnosti u testovima patogenosti na različitim domaćinima familije Cucurbitaceae, kao i na različitim sortama lubenice. Bakteriozna mrljavost plodova lubenice do 2014. godine nije zabeležena u nas...The Cucurbitaceae family consists of over 800 plant species, some of which play an important role in human nutrition, processing and pharmaceutical industries. Bearing in mind these plants are especially susceptible to several types of phytopathogenic bacteria, losses can be substantial in years with favorable conditions for the emergence of infection. In the period 2013‒2016, symptomatic cucurbit plant samples were collected from different locations throughout Serbia. A total of 63 strains of phytopathogenic bacteria were isolated from diseased tissues. The strains were studied using the standard and modern methods. All studied strains were Gram-negative and catalase positive. Out of the 63 strains tested, 61 strains caused the hypersensitivity reaction of tobacco plants. The exceptions were two strains isolated from a watermelon fruit showing the symptoms of bacterial fruit blotch, caused by Acidovorax citrulli. Based on pathogenic, morphological, biochemical-physiological and molecular characteristics, isolated strains were classified into 3 genera: Acidovorax, Pseudomonas and Pectobacterium. Isolated strains, which did not produce potato soft rot, but were oxidase positive and grew at 41˚C, were classified into the genus Acidovorax. They produced beige to tancoloured, round, nonmucoid, convex colonies on yeast extract-dextrose-CaCO3 (YDC) agar. After three days, spreding of the colony margins was noticed, and the colour of the colony became darker. The identity of the strains was also confirmed by using the A. citrulli-specific primer set BX-L1/BX-S-R2. A 279-bp DNA fragment was amplified. The 16S rRNA gene sequence from two strains showed 100% identity to A. citrulli strains, previously deposited in the NCBI GenBank database. Genetic relatedness among strains was investigated by the rep- PCR method. All tested strains, except one, showed the same BOX-PCR profiles. The obtained results show that A. citrulli strains isolated in Serbia belonged to a relatively homogeneous population..

Polyphasic study of phytopathogenic bacterial strains associated with deep bark canker of walnut in Serbia revealed a new species, Brenneria izbisi sp. nov

Serious outbreaks of walnut deep bark canker were observed on young walnut trees (Juglans regia L.) in two localities in the northern part of Serbia during 2020. From the symptomatic walnut tissues, two types of bacterial colonies were isolated, predominantly, light cream, circular and smooth colonies, as well as small, yellowish, mucoid and convex ones. PCR analysis and phenotypic assays suggested that the former group belongs to Brenneria spp., while the latter isolates were identified as Xanthomonas arboricola pv. juglandis. Within the Brenneria group, two strains were identified as Brenneria nigrifluens, while other 15 strains did not belong to any Brenneria species described so far. Therefore, we selected four representative strains of the unknown Brenneria sp. and subjected them to polyphasic analysis. As expected, in a phylogenetic tree based on partial 16S rDNA sequences, four novel strains grouped with other Brenneria representatives, and showed close phylogenetic relationship to Brenneria salicis. Furthermore, multilocus sequence analysis (MLSA) based on the partial sequences of atpD, gyrB, infB and rpoB housekeeping genes and core-genome phylogeny indicated that the studied strains form a novel and a clearly separate Brenneria lineage. Overall genome relatedness indices showed that they represent a new Brenneria species. The new species can be differentiated from the other Brenneria spp. infecting walnut and closely related B. salicis strains based on phenotypic characteristics, as well. Moreover, the pathogenicity tests on two-year-old walnut plants proved the ability of strains to cause necrosis and longitudinal black lesions and cracks on the trunk and branches of walnut trees. Overall, polyphasic characterization showed that the studied strains isolated from walnut with symptoms of deep bark canker represent a novel species of the genus Brenneria for which the name Brenneria izbisi sp. nov. is proposed. The type strain of B. izbisi is KBI 423T (= CFBP 9035T = LMG 32479T). To facilitate rapid identification of newly described species, a conventional PCR protocol and primers targeting the putative gene hrpP, were developed. Further study should reveal the potential role of each pathogen isolated from symptomatic walnut in disease development as well as possible interaction between them

Specificity and sensitivity of three pcr-based methods for detection of erwinia amylovora in pure culture and plant material

Three PCR methods, referred in this study as,conventional" "nested" and,chromosomal" PCR and suggested for routine detection of Erwinia amylovora in pure culture and plant material, were evaluated according to their specificity and sensitivity. Specificity of PCR methods was analyzed by using 42 strains of E. amylovora, originating from different locations and plant species, with diverse PFGE profiles, representing distant populations of the pathogen. Sensitivity of PCR protocols in pure culture was studied by using nine different concentrations of E. amylovora in sterile ultrapure water as a template in PCR reactions. In order to study inhibitory effect of plant DNA and other inhibitors on sensitivity of the three PCR methods bacterial dilutions were mixed with plant macerate of pear, apple and quince prior to the PCR reaction. In specificity assays, tested PCR protocols were able to detect all E. amylovora strains regardless of the host of the strain, its origin or PFGE group, indicating primer specificity. On the other hand, sensitivity among tested methods varied, depending on bacterial concentration and selected plant material used in the PCR. When working with pure cultures nested PCR showed the greatest sensitivity by detecting 1.9 bacterial cells per PCR reaction, followed by detection limit of 9.5 cells per PCR reaction with conventional PCR and 1.9.105 cells/PCR reaction with chromosomal PCR. In spiked samples plant inhibitors either did not affect or they decreased the sensitivity of the PCR reaction, depending on the protocol and/or type of plant macerate. In our experiments, inhibitors from pear and quince macerates did not affect sensitivity of nested PCR, while apple macerate reduced its sensitivity by a factor of 10. Conventional PCR protocol was able to detect 95 cells/PCR reaction in pear and apple macerate, but only 9.5.103 cells/PCR in quince macerate. Greatest decrease in sensitivity of the PCR method was observed in spiked samples with chromosomal PCR since bacterial DNA was not detected in each of the spiked samples. Our research shows that all three PCR protocols are specific for detection of E. amylovora, but nested PCR proved to be most sensitive when working with pure cultures and plant material

Populacija bakterija u bakterioznim tumorima koštičavog voća u Srbiji

Proizvodnja koštičavog voća zauzima značajno mesto u voćarstvu Srbije. Tradicionalno, najviše se gaji šljiva sa sve raznovrsnijim sortimentom u poslednjih desetak godina, dok je proizvodnja trešnje, višnje i kajsije sve više zastupljena u pojedinim regionima zemlje, nekoliko decenija unazad. Prema podacima FAO za 2021. godinu, prinos šljive bio je oko 40.000 t, a trešanja i višanja oko 15.000 t. Iste godine, proizvedeno je oko 30.000 t kajsije. Uspešnu proizvodnju često ometa pojava različitih biljnih bolesti. Od bakterioznih oboljenja, bakteriozni rak korena i korenovog različitih gajenih vrsta zauzima značajno mesto. S obzirom da je utvđeno da bakteriozne tumore osim tumorogenih, naseljavaju i druge vrste bakterija, deo istraživanja posvećen je izučavanju sastava populacije mikroorganizama tumora. Tokom 2020. i 2021. godine na području Srbije, prikupljeni su uzorci šljive, trešnje, višnje i kajsije sa izraženim simptomima bakterioznog raka. Primenom savremene metode sekvenciranja umnoženih produkata regiona V3-V4 16S rRNK gena, dobijene su amplikonske sekvence (ASV), koje su dalje obrađene. Na taj način, determinisane su najzastupljenije vrste bakterija u testiranim uzorcima. Patogena vrsta R. tumorigenes detektovana je kod trećine od ukupnog broja testiranih uzoraka. U uzorcima u kojima je prisutna, njena relativna zastupljenost iznosi do ~ 4%. Rhizobium spp. prisutan je do ~ 11% u uzorcima šljive, odnosno ~ 6% u uzorcima kajsije i ~ 5% trešnje i višnje. Vrste roda Bacillus detektovane su u svim testiranim tumorima, sa udelom i do ~ 53% kod uzoraka šljive. U tumorima na trešnji i višnji, prisutne su vrste roda Rachnella u koncentraciji do ~ 23%, kao i Cutibacterium do ~ 14%. Vrste roda Pseuodmonas detektovane su u uzrocima tumora na kajsiji (do ~4%). Osim navedenih, u uzorcima su prisutni i drugi taksoni u nižim koncentracijama. Predstavljeni rezultati su deo projekta koji se sprovodi u cilju boljeg razumevanja odnosa između patogenih i nepatogenih vrsta u zajednici mikroorganizama tumora. Imajući u vidu sve veći značaj pojave bakterioznog raka u različitim zasadima širom sveta, kao i potencjalni benefit mikroorganizama koji naseljavaju tumore u biološkoj kontroli i opštem stanju biljke domaćina, istraživanja na ovu temu će biti od značaja i u narednom periodu

Bakterioze tikava

Production of cucurbits has a long tradition worldwide. Cucumber, watermelon and melon production is very important for many producers in some parts of Serbia. These crops are especially susceptible to phytopathogenic bacteria which can cause substantial losses in years with favorable weather conditions. In this paper major bacterial diseases of cucurbits are described, such as bacterial fruit blotch caused by Acidovorax citrulli, angular leaf spot caused by Pseudomonas syringae pv. lachrymans, bacterial spot caused by Xanthomonas campestris, bacterial wilt caused by Erwinia tracheiphila, cucurbit yellow wine disease caused by Serratia marcescens, bacterial leaf spot, caused by Pseudomonas syringae pv. syringae, inducing bacterial soft rot caused by Pectobacterium species.Biljke familije Cucurbitaceae imaju dugu tradiciju proizvodnje širom sveta. U pojedinim krajevima Srbije, naročito severnim, proizvodnja krastavca, lubenice i dinje predstavlja primarno zanimanje mnogih poljoprivrednih gazdinstava. Pojava bakterioza u godinama sa uslovima koji pogoduju nastanku infekcije, može značajno ugroziti proizvodnju ovih biljaka. U ovom radu predstavljena su najznačajnija bakteriozna oboljenja biljaka familije Cucurbitaceae, među kojima su: bakteriozna mrljavost plodova lubenice, čiji je prouzrokovač Acidovorax citrulli, uglasta pegavost lišća krastavca, prouzrokovana bakterijom Pseudomonas syringae pv. lachrymans; bakteriozna uvelost krastavca prouzrokovana vrstom Erwinia tracheiphila; bakteriozna pegavost koju prouzrokuje Xanthomonas cucurbitae; potom žutilo vreža, čiji je prouzrokovač Serratia marcescens; zatim bakteriozna lisna pegavost prouzrokovana Pseudomonas syringae pv. syringae; kao i bakteriozna vlažna trulež prouzrokovana vrstama roda Pectobacterium

Draft Genome Sequences of Agrobacterium nepotum Strain 39/7(T) and Agrobacterium sp. Strain KFB 330

Tumorigenic strains of Agrobacterium spp. are responsible for crown gall disease of numerous plant species. We present here draft genome sequences of nonpathogenic Agrobacterium nepotum strain 39/7(T) (CFBP 7436(T), LMG 26435(T)), isolated from crown gall tumor on Prunus cerasifera, and tumorigenic Agrobacterium sp. strain KFB 330 (CFBP 8308, LMG 28674), isolated from galls on raspberry

Novo destruktivno oboljenje krompira - zebrasti čips

Potato production provides one of the main sources for food production, processed either at home or by the industry. Plant diseases are often limiting factor in cultivation of this crop, leading to reduction of yield and tuber quality. Zebra chip disease, caused by a floem-limited bacterium, belonging to the genus Candidatus Liberibacter, is a new, economically important disease of potato and other solanaceous plants. It is widespread in commercial potato fields in USA, Mexico, Central America, New Zealand, and was recently reported in Europe. The disease reduces yield, but most significant damage occurs due to the symptom development on tubers and loss of their marketability. Given the importance of potato production in our country, the goal of this paper is to raise the awareness about the disease occurrence and distribution, discuss the main characteristics of this relatively unknown pathogen, detection and identification methods necessary for the successful and timely diagnosis, epidemiology, symptomatology and disease management.Krompir je biljna vrsta čija proizvodnja ima veoma značajnu ulogu u obezbeđivanju hrane, a takođe i sirovine za industrijsku preradu. Pojava štetnih organizama neretko predstavlja ograničavajući faktor gajenja ove biljke, jer dovodi do smanjenja prinosa i pogoršanja kvaliteta krtola. Oboljenje nazvano zebrasti čips, koje prouzrokuje fastidiozna bakterija iz roda Candidatus Liberibacter, je novo, ekonomski značajno oboljenje biljaka iz familije Solanaceae, čiji je glavni domaćin krompir. Patogen, takođe, zaražava i srodne vrste kao što su paradajz i paprika. Ovo destruktivno oboljenje rasprostranjeno je u proizvodnim područjima Amerike, Novog Zelanda, a nedavno je otkriveno i na evropskom kontinentu. Kao posledica infekcije, dolazi do smanjenja prinosa zaraženih biljaka, dok najznačajnije štete nastaju usled pojave simptoma na krtolama i gubitka njihove tržišne vrednosti. S obzirom na značaj proizvodnje krompira, paradajza i paprike u našoj zemlji, cilj ovog rada je da se ukaže na pojavu i rasprostranjenost oboljenja, glavne karakteristike ovog malo poznatog patogena, metode detekcije i identifikacije neophodne za pouzdanu i pravovremenu dijagnozu oboljenja, epidemiologiju, simptomatologiju i mere zaštite

Bakteriozna oboljenja biljaka familije Cucurbitaceae

Cucurbits production has a long tradition in our country. In some parts of the country, cucumber, watermelon and melon are the most important agricultural crops. High yield and good profit are often compromized by various biotic and abiotic factors. In years with favorable weather conditions cucurbit bacterial diseases can cause serious damage. This paper describes major bacterial diseases of cucurbit plants, such as angular leaf spot caused by Pseudomonas syringae pv. lachrymans, bacterial fruit blotch caused by Acidovorax citrulli, bacterial spot caused by Xanthomonas campestris, bacterial wilt caused by Erwinia tracheiphila and cucurbit yellow wine disease caused by Serratia marcescens, relatively new and invasive disease across the United States. In addition, bacterial leaf spot, caused by Pseudomonas syringae pv. syringae and Pectobacterium carotovorum subsp. carotovorum, inducing bacterial soft rot, are becoming more important in cucurbit production worldwide.Biljke familije Cucurbitaceae se dugi niz godina gaje širom naše zemlje. U pojedinim krajevima, proizvodnja krastavca, lubenice i dinje predstavlja osnovni izvor prihoda za mnoga poljoprivredna domaćinstva. Visok prinos i ekonomsku dobit često ugrožavaju različiti biotski i abiotski faktori. U godinama sa uslovima povoljnim za nastanak infekcije, bakteriozna oboljenja mogu prouzrokovati velike gubitke. U ovom radu predstavljene su najznačajnije bakterioze biljaka familije Cucurbitaceae kao što su uglasta pegavost lišća krastavca, prouzrokovana bakterijom Pseudomonas syringae pv. lachrymans; bakteriozna mrljavost plodova lubenice, čiji je prouzrokovač Acidovorax citrulli; zatim bakteriozna pegavost koju prouzrokuje Xanthomonas cucurbitae; bakteriozna uvelost krastavca prouzrokovana vrstom Erwinia tracheiphila i žutilo vreža, čiji je prouzrokovač Serratia marcescens. Gubicima u proizvodnji doprinose i prouzrokovači bakteriozne pegavosti, Pseudomonas syringae pv. syringae, kao i bakteriozne vlažne truleži, Pectobacterium carotovorum subsp. carotovorum

'Candidatus Liberibacter solanacearum' - prouzrokovač 'zebrastog čipsa' krompira

Candidatus Liberibacter solanacearum (CaLsol) is a phloem-limited pathogen and it was found associated with the 'zebra chip' potato disease. This pathogen causes signifi cant economic losses by reducing the yield and quality of potato crops. It is widespread in commercial potato fi elds in USA, Mexico, Central America ,New Zealand. CaLsol can also affect other solanaceous crops, including tomato, pepper, eggplant, tamarillo. Recently, it was detected in several Northern European countries in carrot and celery crops. Chips made from zebra chip-infected tubers show dark stripes that become more visible upon frying, and are therefore commercially unacceptable. Bearing in mind the importance of potato production in our country, it is necessary to raise the awareness about the disease occurrence and distribution.Među najznačajnijim štetnim organizmima koji ugrožavaju proizvodnju krompira od nedavno se ubraja fastidiozna bakterija 'Candidatus Liberibacter solanacearum' (CaLsol). Ovaj patogen odgovoran je za pojavu novog oboljenja krompira pod nazivom 'zebrasti čips' (ZČ), rasprostranjenog u proizvodnim područjima Amerike i Novog Zelanda. Iako je glavni domaćin patogena krompir, ozbiljne štete zabeležene su i na biljkama paprike, paradajza i drugim gajenim vrstama iz familije Solanaceae. U Evropi isti patogen nedavno je otkriven na obolelim biljkama mrkve i celera. Na krompiru, kao posledica infekcije dolazi do smanjenja prinosa zaraženih biljaka, a najznačajnije štete nastaju usled pojave simptoma na krtolama i gubitka njihove tržišne vrednosti. S obzirom na ekonomski značaj proizvodnje krompira u našoj zemlji, kao i na mogućnost unošenja bakterije putem vegetativnog sadnog materijala, postoji opravdana zabrinutost od širenja ovog patogena na teritoriji naše zemlje

SQUASH AND WATERMELON SOFT ROT CAUSED BY Pectobacterium carotovorum subsp. brasiliense IN SERBIA

The north part of Serbia has a long tradition of growing different varieties of watermelon (Cit­rullus lanatus) and squash (Cucurbitae pepo). Most of the production originates from imported seed material, which creates the risk of introduction of seed-borne pathogens. P. carotovorum strains classified as P. c. subsp. brasiliense have been reported as causal agents of blackleg of potato in 2004 in Brazil. In recent years, the pathogen has spread in many countries on different hosts. In July 2013, fruit macerating symptoms were noticed in two fields of the breeder variety trials in Backi Petrovac. During next season in Celarevo, soft rot symptoms were observed on watermelon vines. Severity of disease was low. White-grey, round, non-mucoid colonies with irregular edges were isolated from symptomatic plant tissue. Three Gram and oxidase negative, catalase positive strains, producing hypersensitive reaction in tobacco leaves were selected for further identification. The strains were facultative anaerobes, non-fluorescent, caused soft rot on potato discs, reduced nitrate and grew at 37 C. They did not produce acid from sorbitol and inositol, nor utilize arginine. The PCR assay was carried out using BR1 f/L 1 r primers specific for P. c. subsp. brasiliense. A specific fragment of 322 bp was amplified for all three strains tested. The 165 rDNA sequence analysis (Gen Bank Accession Nos. MH031784, MH031785, MH031786) showed 99% identity to the previously deposited sequences of P. c. pv. brasiliense. The recA (GenBank Accession Nos. MK911735, MK911736, MK911737) and gapA (GenBank Accession Nos. MK911738, MK911739, MK911740) sequence analysis of three strains showed the highest nucleotide identity (100 - 98.35% and 99.42 - 98.08% respectively), with P. c. pv. brasiliense sequences previously deposited in NCBI Gen Bank database. Pathogenicity of the strains was confirmed on 2-week-old watermelon and melon seedlings and mature zucchini fruits. All test­ed strains induced the symptom development. Bacteria were re-isolated from symptomatic tissues, fulfilling Koch's postulates. Identity of the re-isolated strains was confirmed by PCR assay. This report of P. c. pv. brasiliense causing soft rot of squash and watermelon in Serbia was first published in Plant Disease journal in May 2019. However, Pectobacterium species from cucurbits were not extensively studied in our country, so we assumed that the pathogen was either introduced by contaminated seed recently, or has been present from before but misidentified earlier due to lack of the specific diagnostic tools