Wheat Seed Coating with Streptomyces sp. Strain DEF39 Spores Protects against Fusarium Head Blight.

Streptomycetes are promising candidates for the biological control of Fusarium Head Blight (FHB) in wheat. Studies involving the use of streptomycetes as biological control agents (BCAs) have been limited to the application when the wheat plant is developed, close to the infection on the spike during flowering. Here, we tested the effects of seed treatment with the Streptomyces sp. DEF39 spores before sowing on FHB symptoms' development. The seed treatment protected the plant from infection by Fusarium graminearum by 49% (p = 0.04). We traced Streptomyces sp. DEF39 in plant organs using strain-specific primers here developed, showing that the streptomycete acts as an endophyte, colonizing the plant tissues up to the spike as well as the roots. This work suggests that it is possible to use a streptomycete as a seed coating BCA, able to partially protect wheat from FHB disease.

Grapevine Powdery Mildew: Fungicides for Its Management and Advances in Molecular Detection of Markers Associated with Resistance.

Grapevine powdery mildew is a principal fungal disease of grapevine worldwide. Even though it usually does not cause plant death directly, heavy infections can lead to extensive yield losses, and even low levels of the disease can negatively affect the quality of the wine. Therefore, intensive spraying programs are commonly applied to control the disease, which often leads to the emergence and spread of powdery mildew strains resistant to different fungicides. In this review, we describe major fungicide classes used for grapevine powdery mildew management and the most common single nucleotide mutations in target genes known to confer resistance to different classes of fungicides. We searched the current literature to review the development of novel molecular methods for quick detection and monitoring of resistance to commonly used single-site fungicides against Erysiphe necator. We analyze and compare the developed methods. From our investigation it became evident that this research topic has been strongly neglected and we hope that effective molecular methods will be developed also for resistance monitoring in biotroph pathogens.

Investigating Useful Properties of Four Streptomyces Strains Active against Fusarium graminearum Growth and Deoxynivalenol Production on Wheat Grains by qPCR.

Streptomyces spp. can be exploited as biocontrol agents (BCAs) against plant pathogens such as Fusarium graminearum, the main causal agent of Fusarium head blight (FHB) and against the contamination of grains with deoxynivalenol (DON). In the present research, four Streptomyces strains active against F. graminearum in dual plate assays were characterized for their ability to colonize detached wheat grains in the presence of F. graminearum and to limit DON production. The pathogen and BCA abundance were assessed by a quantitative real-time PCR, while DON production was assessed by HPLC quantification and compared to ergosterol to correlate the toxin production to the amount of fungal mycelium. Fungal growth and mycotoxin production were assessed with both co-inoculation and late inoculation of the BCAs in vitro (three days post-Fusarium inoculation) to test the interaction between the fungus and the bacteria. The level of inhibition of the pathogen and the toxin production were strain-specific. Overall, a higher level of DON inhibition (up to 99%) and a strong reduction in fungal biomass (up to 71%) were achieved when streptomycetes were co-inoculated with the fungus. This research enabled studying the antifungal efficacy of the four Streptomyces strains and monitoring their development in DON-inducing conditions.

Selection of an Endophytic Streptomyces sp. Strain DEF09 From Wheat Roots as a Biocontrol Agent Against Fusarium graminearum.

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.

Evaluation of in-vitro methods to select effective streptomycetes against toxigenic fusaria.

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.

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.

Impact of tricyclazole and azoxystrobin on growth, sporulation and secondary infection of the rice blast fungus, Magnaporthe oryzae.

BACKGROUND: Rice blast, caused by Magnaporthe oryzae B. Couch sp. nov., is one of the most destructive rice diseases worldwide, causing substantial yield losses every year. In Italy, its management is based mainly on the use of two fungicides, azoxystrobin and tricyclazole, that restrain the disease progress. The aim of this study was to investigate and compare the inhibitory effects of the two fungicides on the growth, sporulation and secondary infection of M. oryzae. RESULTS: Magnaporthe oryzae mycelium growth was inhibited at low concentrations of azoxystrobin and relatively high concentrations of tricyclazole, while sporulation was more sensitive to both fungicides and was affected at similarly low doses. Furthermore, infection efficiency of conidia obtained from mycelia exposed to tricyclazole was affected to a higher extent than for conidia produced on azoxystrobin-amended media, even though germination of such conidia was reduced after azoxystrobin treatment. CONCLUSIONS: 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.

Genetic similarity of flag shoot and ascospore subpopulations of Erysiphe necator in Italy.

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 (theta = 0.214 and 0.150, respectively), no differentiation was evident between vineyards in 1998 (theta = 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.