Ecology and characterization of Streptomyces species associated with common scab disease conducive and biofumigated soils in South Africa

Gouws, Reinette (2013-03)

Thesis (PhD(Agric))--Stellenbosch University, 2013.

Thesis

ENGLISH ABSTRACT: Common scab of potato is a serious cosmetic disease in South Africa as well as internationally. The disease affects the appearance and quality of potatoes resulting in major annual losses. Potato producers in South Africa, in the commercial, emerging and processing potato industries, struggle to manage the incidence of common scab, especially soilborne inoculum. Existing products and management programs against common scab are often insufficient. The two main aims of the study were to i) characterize and determine the pathogenic Streptomyces spp. occurring in potato production regions in South Africa and ii) investigate the mechanisms through which Brassica soil amendments can reduce common scab and ways in which it can be included in a sustainable management program. In South Africa, Streptomyces scabiei is still regarded as the main causal agent of common scab. However, world-wide, the disease is caused by a complex of Streptomyces species, with the dominant species varying in different regions. Therefore, a total of 132 Streptomyces isolates collected from six South African potato production regions were characterized. Potato pot trials showed that 53 % of the isolates were pathogenic. Analyses using species specific primers and phylogenetic analyses (16S rRNA phylogeny and multilocus phylogeny) showed that S. scabiei was the most prominent species in South Africa comprising 51.4 % of the pathogenic isolates, followed by S. europascabiei (30 %), S. cavisabies (5.7 %), and S. stelliscabies (1.45 %). The remaining 11.45 % of the pathogenic isolates comprised three taxa, which are related and fit within phylogenetic clades that do not contain common scab isolates from any country other than South Africa. The taxa are named here Streptomyces strains RSA1 (5.7 %), RSA2 (4.3 %) and RSA3 (1.45 %). Streptomyces strain RSA1, which occurred in two production regions, is of special concern since these isolates produce fissure scab symptoms that result in severe cosmetic tuber damage. Fissure scab has not been reported from any other region of the world and is of concern in South Africa since it occurs on the cultivar Mondial that is tolerant to typical common scab. PCR analyses targeting three marker pathogenicity island (PAI) genes (txtAB, nec1, tomA) showed that among the pathogenic isolates nec1 occurred in 89 % of the isolates, tomA in 81 % and txtAB in 89 % of the isolates. The isolates (11 %) that did not contain the txtAB gene and also did not produce thaxtomin, belonged to S. caviscabies and Streptomyces strains RSA2 and RSA3. The incorporation of Brassica tissue into soil has recently shown some potential for reducing common scab disease incidence. Brassica crop residues contain glucosinolates (GLN) that upon cell disruption are hydrolysed by the enzyme myrosinase to yield a diversity of biologically-active hydrolysis products that are toxic to soil microbes. This control mechanism is known as biofumigation. The current study showed that common scab was significantly reduced under field conditions through incorporation of fresh or air-dried residues of Brassica oleracea var. capitata (cabbage) in two consecutive potato plantings. The in-vitro effect of volatile emissions from various Brassica species towards Streptomyces was evaluated using two bioassay methods. An in-vitro agar plate bioassay showed that, in general, volatile emissions from water activated freeze-dried tissue of a B. juncea/S. alba mix and B. napus were superior to those from B. oleracea var italica and B. oleracea var capitata for suppression of growth and sporulation of Streptomyces. In a gas chamber bioassay that used freshly macerated Brassica tissue, B. oleracea var capitata and a B. juncea/S. alba mix suppressed sporulation but not hyphal growth of Streptomyces. The gas chamber bioassay showed that the biofumigation effect was bacteriostatic, i.e. isolates recovered after volatile exposure. Both bioassays showed that significant components of both the pathogenic (50 %) and non-pathogenic (20 %) Streptomyces population examined were unaffected by the Brassica tissue derived volatiles. Mechanisms of disease reduction through Brassica amendments are not limited to biofumigation, but changes in the structure of microbial communities involved in systemic induced resistance and/or general microbial suppression may also contribute to disease suppression. In the current study a potato split-root experiment that spatially separated the progeny tubers and roots of Brassica juncea/ Sinapus alba (mustard mix) and Brassica oleracea var oleracea (cabbage) amended soil sub-units from non-amended soil sub-units, showed that induced resistance induced in plants was involved in common scab suppression. The role of toxic GLN hydrolysis products was ruled out in the induced resistance mediated disease suppression, since volatiles were released from Brassica amended soil prior to initiating the experiment. Increased microbial activity in the Brassica amended units was evidenced by significant increases in ß-glucosidase and urease activities. Principal component analyses revealed some trends in the overall soil, tuber and root associated microbial genera (Trichoderma, Pseudomonas, Streptomyces, total bacteria and Fusarium) in the Brassica amended and non-amended units. The mustard amended treatment, and to a lesser extend the cabbage amended units, showed trends towards increases in soil Fusarium and Trichoderma and root Trichoderma populations, and decreases in total bacterial and Streptomyces populations in soil and tubers, and Streptomyces in roots. This study has contributed towards our knowledge of the Streptomyces species causing potato common scab in South Africa, and mechanisms through which Brassica soil amendments can reduce common scab. Several Streptomyces species, including novel pathogenic taxa, are involved in causing common scab and their differential virulence, and responses to being suppressed by Brassica amendments will require the implementation of an integrated management program. The planting of cabbage as a cash crop, with the subsequent incorporation of residues into soil shows promise as a management strategy for subsistence farmers. The mechanisms involved in common scab suppression through Brassica amendments were shown to involve systemic induced resistance in plants and general microbial suppression. Altogether, knowledge obtained in this study can be used to i) optimize management strategies for sustainable potato production, ii) further elucidate the mechanisms involved in disease suppression and iii) develop molecular techniques, such as quantitative real-time PCR for rapid identification and quantification of common scab-causing species in South Africa.

AFRIKAANSE OPSOMMING: Bruinskurf is ‘n ernstige kosmetiese siekte op aartappels in Suid Afrika sowel as internasionaal. Die siekte affekteer die voorkoms en kwaliteit van aartappels en lei dikwels tot aansienlike jaarlikse verliese. Aartappel produsente in Suid Afrika, insluitend kommersiële boere, die opkomende landbou sektor en die verwerkings bedryf, vind dit moeilik om bruinskurf voorkoms te bestuur, veral die grondgedraagde inokulum. Bestaande produkte en bestuursprogramme teen bruinskurf is nie voldoende nie. Die twee hoofdoelwitte van hierdie studie was om i) die patogeniese Streptomyces spp. in aartappel produksie streke te karakteriseer en ii) die meganismes waardeur die inkorporering van Brassica reste in die grond bruinskurf voorkoms kan verminder, te bestudeer en wyses te vind waarop dit ingesluit kan word in ‘n onderhoubare bestuursprogram. Streptomyces scabiei word steeds beskou as die hoofveroorsakende agent vir bruinskurf in Suid Afrika. Wêreldwyd word die siekte egter veroorsaak deur ‘n kompleks van Streptomyces spesies met die dominante spesie wat verskil in elke produksie area. In die lig hiervan is a totaal van 132 Streptomyces isolate vanaf ses produksie areas in Suid Afrika versamel en gekarakteriseer. Aartappel potproewe het getoon dat 53% van hierdie isolate patogenies was. Deur gebruik te maak van spesie spesifieke “primers” en filogenetiese analise (16S rRNA filogenie en multilokus filogenie) is getoon dat S. scabiei die mees prominente spesie in Suid Afrika is met 51.4% van die patogeniese isolate wat positief getoets het vir hierdie spesie, gevolg deur S. europaeiscabiei (30%), S. caviscabies (5.7%), and S. stelliscabies (1.45 %). Die oorblywende 11.45 % van die patogeniese isolate bestaan uit drie taksa wat verwant is en inpas binne filogenetiese “clades” wat nie bruinskurf isolate van enige land behalwe Suid Afrika bevat nie. Die taksa word hier genoem Streptomyces taxa RSA1 (5.7 %), RSA2 (4.3 %) en RSA3 (1.45 %). Streptomyces taxon RSA1, wat voorgekom het in twee produksie areas, is van spesifieke belang omdat hierdie isolate spleetskurf simptome produseer wat lei tot aansienlike kosmetiese knolskade. Spleetskurf is nog nie in enige ander produksie streek in die wêreld gerapporteer nie en is veral van groot belang in Suid Afrika omdat dit op die bruinskurf tolerante kultivar, Mondial, voorkom. PKR analises wat die drie merker patogenisiteit eiland (PAE) gene (txtAB, nec1, tomA) teiken, het getoon dat nec1 89 %, tomA, 81 % en txtAB 89 % voorgekom het in die patogeniese isolate. Die isolate (11 %) wat nie die txtAB gene bevat het nie en ook nie thaxtomin A produseer het nie, het behoort aan S. caviscabies en Streptomyces taxa RSA2 en RSA3 groepe. Die inkorporering van Brassica reste in die grond het onlangs potentiaal getoon om bruinskurf siekte voorkoms te verminder. Brassica reste bevat glukosinolate (GLN) wat tydens selvernietiging gehidroliseer word deur die ensiem mirosinase, om ‘n diverse groep biologiese aktiewe hidrolise produkte te lewer wat toksies is vir grond mikrobe. Hierdie meganisme van beheer staan bekend as bioberoking. Die huidige studie het getoon dat bruinskurf voorkoms betekenisvol verminder is deur die inkorporering van vars en lugdroog Brassica oleracea var. capitata (kopkool) reste onder veld toestande in twee opeenvolgende aartappel aanplantings. Die effek van vlugtige verbindings vanaf verskeie Brassica spesies op Streptomyces is geevalueer deur middel van twee in-vitro bio-analise tegnieke. ‘n In-vitro agarplaat bio-analise het getoon dat vlugtige verbindings van water-geaktiveerde vriesdroog reste van ‘n B. juncea / S. alba mengsel en B. napus oor die algemeen meer effektief was om Streptomyces groei en sporulasie te onderdruk as B. oleracea var italica en B. oleracea var. capitata reste. In ‘n gaskamer bio-analise waar daar gebruik gemaak is van varsgemaalde Brassica reste het B. oleracea var. capitata en ‘n B. juncea / S. alba mengsel die sporulasie van Streptomyces onderdruk maar nie die hifegroei nie. Die gaskamer bio-analise het getoon dat die bioberoking effek bakteriostaties is omdat die isolaat groei herstel het na blootstelling aan die vlugtige verbindings. Beide bio-analises het getoon dat betekenisvolle komponente van beide die patogeniese (50 %) en nie-patogeniese (20 %) Streptomyces populasies wat ondersoek is glad nie deur die Brassica-reste geinduseerde vlugtige verbindings geaffekteer is nie. Meganismes van siekte vemindering deur Brassica inkorporering is nie beperk tot bioberoking nie, maar veranderings in die struktuur van mikrobiese gemeenskappe betrokke in ge-induseerde sistemiese weerstand (GSW) en/of algemene onderdrukking kan ook bydra tot siekte onderdrukking. In die hierdie studie het ‘n aartappel wortel-split eksperiment waar die nageslagknolle en wortels ruimtelik geskei is in subeenhede wat gevul was met B. juncea / S. alba (mosterd mengsel) of B. oleracea var oleracea (kopkool) behandelde of nie-behandelde grond, getoon dat sistemiese onderdrukking betrokke is in bruinskurf siekte voorkoms. Die rol van toksiese GLN hidrolise produkte is uitgeskakel in hierdie siekte onderdrukking omdat die vlugtige verbindings voor grond inkorporering vrygestel is uit die Brassica weefsel. Verhoogde mikrobiese aktiwiteit in die Brassica behandelde subeenhede was bevestig deur betekenisvolle verhogings in β-glukosidase en urease aktiwiteit. “Principle component” analise het sekere tendense uitgelig in die algehele grond, knol en wortel-geassosieerde mikrobiese genera (Trichoderma, Pseudomonas, Streptomyces, totale bakterieë en Fusarium) in die Brassica behandelde en onbehandelde subeenhede. Die mosterd behandelde, en tot ‘n mindere mate, die kool behandelde eenhede, het getoon dat daar ‘n verhoging in grond Fusarium en Trichoderma en wortel Trichoderma populasies en ‘n afname in totale bakterieë en Streptomyces populasies in grond en knolle en Streptomyces populasies in wortels was. Hierdie studie het bygedra tot ons kennis oor Streptomyces spesies wat bruinskurf op aartappels veroorsaak in Suid Afrika en meganismes waardeur inkorporering van Brassica materiaal in die grond bruinskurf kan verminder. Verskeie Streptomyces spesies, insluitend moontlike nuwe patogeniese spesies, is betrokke by bruinskurf voorkoms en hul onderskeie virulensies en reaksie op die inkorporering van Brassica materiaal vereis die implementering van ‘n geïntegreerde bestuursprogram. Die plant van kopkool as ‘n kontant gewas met gevolglike inkorporering van koolreste hou belofte in as ‘n bestuurs strategie vir die opkomende landbou sektor. Die meganismes betrokke in bruinskurf onderdrukking deur Brassica toevoeging in grond het getoon dat plant geinduseerde sistemiese weerstand sowel as algemene onderdrukking ‘n rol speel. Altesaam kan die kennis wat deur hierdie studie ingewin is gebruik word om i) volhoubare aartappel produksie stelsels te optimiseer, ii) meganismes betrokke by siekte onderdrukking verder te verken en iii) molekulêre tegnieke soos “real-time” PKR te ontwikkel vir spoedige identifikasie en kwantifisering van bruinskurf veroorsakende spesies in Suid Afrika.

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