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.

This study presents for the first time detailed azoxystrobin and tricyclazole growth-response curves for M. oryzae mycelium growth and sporulation. Furthermore, high efficacy of tricyclazole towards inhibition of sporulation and secondary infection indicates an additional possible mode of action of this fungicide that is different from inhibition of melanin biosynthesis.

BackgroundIn 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.ResultsThe 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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0886-1) contains supplementary material, which is available to authorized users.

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. (106 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.

The overwintering mode of the grape powdery mildew fungus, Erysiphe necator (syn. Uncinula necator), as mycelium in dormant buds (resulting in symptoms known as flag shoots) or as ascospores in cleistothecia, affects the temporal dynamics of epidemics early in the growing season. We tested whether distinct genetic groups (I and III) identified previously in E. necator correlate to overwintering modes in two vineyards in Tuscany, Italy, to determine whether diagnostic genetic markers could be used to predict overwintering. Samples from one vineyard were collected from flag shoots; the other vineyard, 60 km away, had no flag shoots, and mildew colonies were assumed to be derived from ascospores. Genetic markers putatively diagnostic for groups I and III showed that both types were common in the flag shoot subpopulation. Both genetic types were found in the ascospore population, although group III was dominant. We did not find strong genetic differentiation between the two subpopulations based on inter-simple sequence repeat markers. Although there was significant (P < 0.001) genetic differentiation between these subpopulations in 1997 and when 1997 and 1998 subpopulations were pooled ( ‫؍‬ 0.214 and 0.150, respectively), no differentiation was evident between vineyards in 1998 ( ‫؍‬ 0.138, P ‫؍‬ 0.872). Moreover, we did not observe distinct lineages corresponding to overwintering modes, as observed in previous studies. We could not determine if differentiation resulted from biological differences or restricted gene flow between the two vineyards. Our samples were taken from both subpopulations early in the epidemic, while previous studies confounded overwintering mode and sampling time. These results do not support a strong correlation between overwintering and genetic groups, highlighting the need to base population biology studies on sound biological and epidemiological knowledge.

Selection of biological control agents (BCA) profits from an integrated study of the tripartite interactions occurring among the BCA, the plant and the pathogen. The environment plays a crucial role in the efficacy of BCA, therefore, the selection process shall utmost mimic naturally occurring conditions. To identify effective biocontrol strains against Fusarium graminearum, the major cause of Fusarium head blight (FHB) in wheat and deoxynivalenol (DON) accumulation in grains, a workflow consisting of in vitro and in vivo assays was set up. Twenty-one Streptomyces strains, 16 of which were endophytes of different plants, were analyzed. In vitro and in vivo tests characterized their plant growth promoting (PGP) traits. Biocontrol activity against F. graminearum was firstly assessed with a dual culture assay. An in vivo germination blotter assay measured Fusarium foot rot and root rot symptoms (FFR-FRR) reduction as well as growth parameters of the plant treated with the Streptomyces strains. A selected subset of Streptomyces spp. strains was then assessed in a growth chamber measuring FFR symptoms and growth parameters of the wheat plant. The approach led to the identification of an effective Streptomyces sp. strain, DEF09, able to inhibit FHB on wheat in controlled conditions by blocking the spread of the pathogen at the infection site. The results were further confirmed in field conditions on both bread and durum wheat, where DEF09 decreased disease severity up to 60%. This work confirms that FRR and FFR pathosystems can be used to identify BCA effective against FHB.

Biocontrol microorganisms are emerging as an effective alternative to pesticides. Ideally, biocontrol agents (BCAs) for the control of fungal plant pathogens should be selected by an in vitro method that is high-throughput and is predictive of in planta efficacy, possibly considering environmental factors, and the natural diversity of the pathogen. The purpose of our study was (1) to assess the effects of Fusarium strain diversity (N = 5) and culture media (N = 6) on the identification of biological control activity of Streptomyces strains (N = 20) against Fusarium pathogens of wheat in vitro and (2) to verify the ability of our in vitro screening methods to simulate the activity in planta. Our results indicate that culture media, Fusarium strain diversity, and their interactions affect the results of an in vitro selection by dual culture assay. The results obtained on the wheat-based culture media resulted in the highest correlation score (r = 0.5) with the in planta root rot (RR) inhibition, suggesting that this in vitro method was the best predictor of in planta performance of streptomycetes against Fusarium RR of wheat assessed as extension of the necrosis on the root. Contrarily, none of the in vitro plate assays using the media tested could appropriately predict the activity of the streptomycetes against Fusarium foot rot symptoms estimated as the necrosis at the crown level. Considering overall data of correlation, the activity in planta cannot be effectively predicted by dual culture plate studies, therefore improved in vitro methods are needed to better mimic the activity of biocontrol strains in natural conditions. This work contributes to setting up laboratory standards for preliminary screening assays of Streptomyces BCAs against fungal pathogens.

Kunova, A., Pizzatti, C., Bonaldi, M., and Cortesi, P. 2014. Sensitivity of nonexposed and exposed populations of Magnaporthe oryzae from rice to tricyclazole and azoxystrobin. Plant Dis. 98:512-518.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 [ED 50 ] = 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 ED 50 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.