Selection of Streptomyces against soil borne fungal pathogens by a standardized dual culture assay and evaluation of their effects on seed germination and plant growth.

BACKGROUND: In the search for new natural resources for crop protection, streptomycetes are gaining interest in agriculture as plant growth promoting bacteria and/or biological control agents. Because of their peculiar life cycle, in which the production of secondary metabolites is synchronized with the development of aerial hyphae and sporulation, the commonly used methods to screen for bacterial antagonists need to be adapted. RESULTS: The dual culture assay was standardized in terms of inoculation timing of Streptomyces antagonist and pathogen, and growth rate of different fungal pathogens. In case of fast-growing fungi, inoculation of the antagonist 2 or 3 days prior to the pathogen resulted in significantly stronger inhibition of mycelium growth. One hundred and thirty Streptomyces strains were evaluated against six destructive soil borne pathogens. The activity of strains varied from broad-spectrum to highly specific inhibition of individual pathogens. All strains inhibited at least one tested pathogen. Three strains, which combined the largest broad-spectrum with the highest inhibition activity, were selected for further characterization with four vegetable species. All of them were able to colonize seed surface of all tested vegetable crops. They mostly improved radicle and hypocotyl growth in vitro, although no statistically significant enhancement of biomass weight was observed in vivo. Occasionally, transient negative effects on germination and plant growth were observed. CONCLUSIONS: The adapted dual culture assay allowed us to compare the inhibition of individual Streptomyces strains against six fungal soil borne pathogens. The best selected strains were able to colonize the four vegetable crops and have a potential to be developed into biocontrol products. Although they occasionally negatively influenced plant growth, these effects did not persist during the further development. Additional in vivo studies are needed to confirm their potential as biological control or plant growth promoting agents.

Biological Control of Lettuce Drop and Host Plant Colonization by Rhizospheric and Endophytic Streptomycetes.

Lettuce drop, caused by the soil borne pathogen Sclerotinia sclerotiorum, is one of the most common and serious diseases of lettuce worldwide. Increased concerns about the side effects of chemical pesticides have resulted in greater interest in developing biocontrol strategies against S. sclerotiorum. However, relatively little is known about the mechanisms of Streptomyces spp. as biological control agents against S. sclerotiorum on lettuce. Two Streptomyces isolates, S. exfoliatus FT05W and S. cyaneus ZEA17I, inhibit mycelial growth of Sclerotinia sclerotiorum by more than 75% in vitro. We evaluated their biocontrol activity against S. sclerotiorum in vivo, and compared them to Streptomyces lydicus WYEC 108, isolated from Actinovate®. When Streptomyces spp. (10(6) CFU/mL) were applied to S. sclerotiorum inoculated substrate in a growth chamber 1 week prior lettuce sowing, they significantly reduced the risk of lettuce drop disease, compared to the inoculated control. Interestingly, under field conditions, S. exfoliatus FT05W and S. cyaneus ZEA17I protected lettuce from drop by 40 and 10% respectively, whereas S. lydicus WYEC 108 did not show any protection. We further labeled S. exfoliatus FT05W and S. cyaneus ZEA17I with the enhanced GFP (EGFP) marker to investigate their rhizosphere competence and ability to colonize lettuce roots using confocal laser scanning microscopy (CLSM). The abundant colonization of young lettuce seedlings by both strains demonstrated Streptomyces' capability to interact with the host from early stages of seed germination and root development. Moreover, the two strains were detected also on 2-week-old roots, indicating their potential of long-term interactions with lettuce. Additionally, scanning electron microscopy (SEM) observations showed EGFP-S. exfoliatus FT05W endophytic colonization of lettuce root cortex tissues. Finally, we determined its viability and persistence in the rhizosphere and endorhiza up to 3 weeks by quantifying its concentration in these compartments. Based on these results we conclude that S. exfoliatus FT05W has high potential to be exploited in agriculture for managing soil borne diseases barely controlled by available plant protection products.

Metrafenone resistance in a population of Erysiphe necator in northern Italy.

BACKGROUND: Metrafenone has been used in Europe in integrated pest management programmes since 2006 to control powdery mildews, including Erysiphe necator. Its exact mode of action is not known, but it is unique among fungicide classes used in powdery mildew management. Recently, resistance to metrafenone was reported in Blumeria graminis f. sp. tritici. In this study we investigated metrafenone resistance in Erysiphe necator in northern Italy. RESULTS: Metrafenone efficacy to control grapevine powdery mildew was monitored in three consecutive years in the field, and its reduced activity was observed in 2013. Out of 13 monoconidial isolates, two sensitive strains were identified, which did not grow at the fungicide concentration recommended for field application. The remaining strains showed variable response to metrafenone, and five of them grew and sporulated similarly to the control, even at 1250 mg L(-1) of metrafenone. Moreover, the resistant strains showed cross-resistance to pyriofenone, which belongs to the same FRAC group as metrafenone. CONCLUSION: The results indicate the emergence of metrafenone resistance in an Italian population of Erysiphe necator. Further studies are needed to gain insight into the metrafenone's mode of action and to understand the impact of resistance on changes in the pathogen population structure, fitness and spread of resistant strains, which will be indicative for designing appropriate antiresistance measures.

Colonization of lettuce rhizosphere and roots by tagged Streptomyces.

Beneficial microorganisms are increasingly used in agriculture, but their efficacy often fails due to limited knowledge of their interactions with plants and other microorganisms present in rhizosphere. We studied spatio-temporal colonization dynamics of lettuce roots and rhizosphere by genetically modified Streptomyces spp. Five Streptomyces strains, strongly inhibiting in vitro the major soil-borne pathogen of horticultural crops, Sclerotinia sclerotiorum, were transformed with pIJ8641 plasmid harboring an enhanced green fluorescent protein marker and resistance to apramycin. The fitness of transformants was compared to the wild-type strains and all of them grew and sporulated at similar rates and retained the production of enzymes and selected secondary metabolites as well as in vitro inhibition of S. sclerotiorum. The tagged ZEA17I strain was selected to study the dynamics of lettuce roots and rhizosphere colonization in non-sterile growth substrate. The transformed strain was able to colonize soil, developing roots, and rhizosphere. When the strain was inoculated directly on the growth substrate, significantly more t-ZEA17I was re-isolated both from the rhizosphere and the roots when compared to the amount obtained after seed coating. The re-isolation from the rhizosphere and the inner tissues of surface-sterilized lettuce roots demonstrated that t-ZEA17I is both rhizospheric and endophytic.

Sensitivity of Nonexposed and Exposed Populations of Magnaporthe oryzae from Rice to Tricyclazole and Azoxystrobin.

Magnaporthe oryzae is the major pathogen of cultivated rice worldwide, which can cause substantial losses to rice production. Rice blast management is based predominantly on the application of fungicides; however, only a little is known about responses of pathogen populations to the most widely used fungicides. In this work, the baseline sensitivity of the Italian M. oryzae population to tricyclazole and azoxystrobin in terms of mycelium growth was determined, and the possible adaptation of the pathogen population after several years of repeated exposure to fungicide treatments was evaluated. All the analyzed strains demonstrated higher sensitivity and variability to azoxystrobin (concentration of fungicide causing 50% growth inhibition [ED50] = 0.063 mg liter-1) than to tricyclazole (99.289 mg liter-1). After comparing two additional populations collected from fields repeatedly treated with fungicides to the baseline, no decrease in sensitivity toward these fungicides was observed and no resistant strains were detected. The shift of the pathogen sensitivity toward these fungicides has not occurred, although we observed slightly increased variance associated with ED50 of azoxystrobin. Therefore, both azoxystrobin and tricyclazole can be used to manage rice blast in Italy but it will be important to continue monitoring M. oryzae population to early detect possible azoxystrobin resistance.