Rapid and Easy Modified Plate-based Screening Methods for Quantitative and Qualitative Detection of Protease Production by Fungi

Proteases constitute a significant part of cell wall-degrading enzymes (CWDEs) produced by fungal biocontrol agents and particularly crucial in mycoparasitism of fungal phytopathogens. Plate-based screening methods are routinely used for screening protease-producing microorganisms including fungi. Skim milk agar (SMA) is one of the most popular media for the detection of protease producing bacteria. However, SMA is not efficient to test fast growing fungi, because it does not give an estimation of the actual amount of secreted protease produced by fungal inocula. In the current study, the efficacy of two modified plate-screening methods, including split-SMA (SSMA) and minimal medium supplemented with skim milk (MSMW) was assessed for detection of protease production by three representative fungal strains including Trichoderma longibrachiatum strain N, Beauveria bassiana strain B and Purpureocillium lilacinum strain PL. Protease production was revealed on the three tested media by the three strains. However, the halo diameter of the fungal strains (a proxy for protease production) was the smallest on SMA. Furthermore, protease production could not be detected for T. longibrachiatum strain N on SMA due to its fast growth; while it showed the highest protease activity on both modified media compared with the other strains. According to the result of this study, the SSMA medium is an easy and more accurate method compared with the two other different methods as it displays the actual amount of protease produced by fungal strains and therefore this method is recommended for quantitative and qualitative detection of protease production by slow and fast growing fungi

The perception of indole negatively modulates biocontrol activities in the plant beneficial Rhizobacterium Lysobacter capsici AZ78

The rhizosphere is a dynamic environment characterised by multiple and complex microbial interactions where diffusible communication signals (DCS) continuously influence the expression patterns of the microbiome, hence regulating fundamental traits for adaptation to the rhizosphere. In particular, plant-associated bacteria release indole, a Volatile Organic Compound (VOC) that acts as an interkingdom signal able to influence antibiotic resistance, motility, biofilm formation and virulence. Lysobacter spp. are commonly found in the rhizosphere and have been frequently associated to disease suppression. For instance, the biocontrol activity of the plant beneficial bacterium Lysobacter capsici AZ78 (AZ78) has been reported against the phytopathogenic oomycetes Phytophthora infestans, Plasmopara viticola, Pythium ultimum and the Gram-positive bacterium Rhodococcus fascians. However, there is scarce information about Lysobacter spp. ecology and how DCS, and in particular indole, may affect their behaviour in the rhizosphere. To investigate the aspects determining rhizosphere competence and functioning of Lysobacter spp., this work presents a functional and transcriptomic analysis performed on AZ78, which was grown in the presence indole. The presence of indole significantly reduced the inhibition capacity of AZ78 against P. ultimum and R. fascians by 47 and 31%, respectively. Moreover, RNA-Seq analysis revealed that nearly 12% of all genes in AZ78 genome were modulated by indole. In particular, indole downregulated the expression of the heat-stable antifungal factor (HSAF) biosynthetic gene cluster, which may affect AZ78 antioomycete and antimicrobial activity. Moreover, in the presence of indole, AZ78 downregulated several signal transduction pathways responsible for nutrients uptake, resulting in reduced growth. Finally, indole downregulated several genes related to type IV pilus functionality, which might lead to impaired twitching motility. This study sheds light on the key role of DCS such as indole in shaping AZ78 behaviour in the rhizosphere and suggests that, manipulating DCS levels may alter the persistence and functioning of several plant-beneficial rhizobacteria, such as Lysobacter strains

Key impact of an uncommon plasmid on bacillus amyloliquefaciens subsp. plantarum S499 developmental traits and lipopeptide production

The rhizobacterium Bacillus amyloliquefaciens subsp. plantarum S499 (S499) is particularly efficient in terms of the production of cyclic lipopeptides, which are responsible for the high level of plant disease protection provided by this strain. Sequencing of the S499 genome has highlighted genetic differences and similarities with the closely related rhizobacterium B. amyloliquefaciens subsp. plantarum FZB42 (FZB42). More specifically, a rare 8008 bp plasmid (pS499) harboring a rap-phr cassette constitutes a major distinctive element between S499 and FZB42. By curing this plasmid, we demonstrated that its presence is crucial for preserving the typical physiology of S499 cells. Indeed, the growth rate and extracellular proteolytic activity were significantly affected in the cured strain (S499 P-). Furthermore, pS499 made a significant contribution to the regulation of cyclic lipopeptide production. Surfactins and fengycins were produced in higher quantities by S499 P-, whereas lower amounts of iturins were detected. In line with the increase in surfactin release, bacterial motility improved after curing, whereas the ability to form biofilm was reduced in vitro. The antagonistic effect against phytopathogenic fungi was also limited for S499 P-, most probably due to the reduction of iturin production. With the exception of this last aspect, S499 P- behavior fell between that of S499 and FZB42, suggesting a role for the plasmid in shaping some of the phenotypic differences observed in the two strains. © 2017 Molinatto, Franzil, Steels, Puopolo, Pertot and Ongena

Susceptibility to Armillaria mellea root rot in grapevine rootstocks commonly grafted onto Teroldego Rotaliano

Armillaria root rot is an increasing problem in some grapevine-growing areas in north-eastern Italy (Trentino Province). The susceptibility of seven grapevine rootstocks (Schwarzmann, 3309 C, 101-14, Teleki 5C, SO4, Kober 5BB and 41 B; all grafted with Teroldego Rotaliano) to Armillaria mellea was evaluated in a five-year investigation. Two inoculation methods were also compared: young grapevine plants were transplanted to a substrate that had been inoculated with A. mellea (Method A), or A. mellea rhizomorphs were inserted under the root bark after the root bark had been lifted up with a scalpel (Method B). Plants inoculated with Method A had higher infection and mortality rates than plants that were inoculated with Method B, demonstrating that root wounding does not lead to higher A. mellea infection. The significantly higher mortality and infection rates of 3309 C as compared with Teleki 5C in the final year of the study suggest that a Teroldego Rotaliano vineyard established on 3309 C will suffer greater losses than would a similar vineyard established on Teleki 5C. Rootstocks that were intermediate in their response to infection (Schwarzmann, Kober 5BB, and 41B) may offer moderate levels of resistance since with these rootstocks the mortality and infection rates were not signifi cantly different from those of Teleki 5C. Since all rootstocks became infected, however, no rootstock is completely immune

Humic acid enhances the growth of tomato promoted by endophytic bacterial strains through the activation of hormone-, growth-, and transcription-related processes

Plant growth-promoting bacteria (PGPB) are promising alternatives in the reduction of the use of chemical fertilizers. Likewise, humic acid (HA) can improve plant growth and/or the establishment of endophytic PGPB. Although the effects of PGPB colonization or HA treatment have been studied separately, little information is available on plant response to the combined applications of PGPB and HA. Thus, the aim of this work was to understand the physiological effects, bacterial colonization and transcriptional responses activated by endophytic bacterial strains in tomato roots and shoots in the absence (control condition) and presence of HA (HA condition). Tomato shoot length was promoted by seed inoculation with Paraburkholderia phytofirmans PsJN, Pantoea agglomerans D7G, or Enterobacter sp. 32A in the presence of HA, indicating a possible complementation of PGPB and HA effects. Tomato colonization by endophytic bacterial strains was comparable in the control and HA condition. The main transcriptional regulations occurred in tomato roots and the majority of differentially expressed genes (DEGs) was upregulated by endophytic bacterial strains in the HA condition. Half of the DEGs was modulated by two or three strains as possible common reactions to endophytic bacterial strains, involving protein metabolism, transcription, transport, signal transduction, and defense. Moreover, strain-specific tomato responses included the upregulation of signal transduction, transcription, hormone metabolism, protein metabolism, secondary metabolism, and defense processes, highlighting specific traits of the endophyte-tomato interaction. The presence of HA enhanced the upregulation of genes related to signal transduction, hormone metabolism, transcription, protein metabolism, transport, defense, and growth-related processes in terms of number of involved genes and fold change values. This study provides detailed information on HA-dependent enhancement of growth-related processes stimulated by endophytic bacterial strains in tomato plants and reports the optimized dosages, complementation properties and gene markers for the further development of efficient PGPB- and HA-based biostimulant

Plasmopara viticola infection affects mineral elements allocation and distribution in Vitis vinifera leaves

Plasmopara viticola is one of the most important pathogens infecting Vitis vinifera plants. The interactions among P. viticola and both susceptible and resistant grapevine plants have been extensively characterised, at transcriptomic, proteomic and metabolomic levels. However, the involvement of plants ionome in the response against the pathogen has been completely neglected so far. Therefore, this study was aimed at investigating the possible role of leaf ionomic modulation during compatible and incompatible interactions between P. viticola and grapevine plants. In susceptible cultivars, a dramatic redistribution of mineral elements has been observed, thus uncovering a possible role for mineral nutrients in the response against pathogens. On the contrary, the resistant cultivars did not present substantial rearrangement of mineral elements at leaf level, except for manganese (Mn) and iron (Fe). This might demonstrate that, resistant cultivars, albeit expressing the resistance gene, still exploit a pathogen response mechanism based on the local increase in the concentration of microelements, which are involved in the synthesis of secondary metabolites and reactive oxygen species. Moreover, these data also highlight the link between the mineral nutrition and plants\u2019 response to pathogens, further stressing that appropriate fertilization strategies can be fundamental for the expression of response mechanisms against pathogens

A critical review of plant protection tools for reducing pesticide use on grapevine and new perspectives for the implementation of IPM in viticulture

Several pests and diseases have grapevine as their favourite host and the vineyard as preferred environment, so an intensive pesticide schedule is usually required to meet qualitative and quantitative production standards. The need to prevent the negative impact of synthetic chemical pesticides on human health and the environment and the consumer expectations in term of chemical residues in food stimulated the research of innovative tools and methods for sustainable pest management. The research project PURE (www.pure-ipm.eu) was a Europe-wide framework, which demonstrated that several solutions are now available for the growers and evaluated several new alternatives that are under development or almost ready for being applied in practice. Although the use of resistant/tolerant varieties is not yet feasible in several traditional grape growing areas, at least part of the synthetic chemical pesticides can be substituted with biocontrol agents to control pests and pathogens and/or pheromone mating disruption, or the number of treatments can be reduced by the use of decision support systems, which identify the optimal timing for the applications. This review presents the state of the art and the perspectives in the field of grapevine protection tools and strategies

Versatile transporter apparatus for experiments with optically trapped Bose-Einstein condensates

We describe a versatile and simple scheme for producing magnetically and optically-trapped Rb-87 Bose-Einstein condensates, based on a moving-coil transporter apparatus. The apparatus features a TOP trap that incorporates the movable quadrupole coils used for magneto-optical trapping and long-distance magnetic transport of atomic clouds. As a stand-alone device, this trap allows for the stable production of condensates containing up to one million atoms. In combination with an optical dipole trap, the TOP trap acts as a funnel for efficient loading, after which the quadrupole coils can be retracted, thereby maximizing optical access. The robustness of this scheme is illustrated by realizing the superfluid-to-Mott insulator transition in a three-dimensional optical lattice

Replacement of Contentious Inputs in Organic Farming Systems (RELACS) – a comprehensive Horizon 2020 project

Organic farmers adhere to high standards in producing quality food while protecting the environment. However, organic farming needs to improve continuously to keep meeting its ambitious objectives. The project ‘Replacement of Contentious Inputs in Organic Farming Systems’ (RELACS) will foster the development and adoption of cost-efficient and environmentally safe tools and technologies to further reduce the use of external inputs on organic farms across Europe as well as in Non EU Mediterranean countries. Project partners will provide scientific support to develop fair and implementable EU rules to improve current practices in organic farming. Farm advisory networks in 11 European countries will reach out to farmers to ensure effective dissemination and adoption of the tools and techniques