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

A review of knowledge on the mechanisms of action of the rare sugar D‐tagatose against phytopathogenic oomycetes

d-tagatose is a rare monosaccharide, naturally present at low concentrations in some fruits and dairy products. d-tagatose is "generally recognized as safe" and is used as a low-calorie sweetener in the food industry. It is able to inhibit the growth of numerous microorganisms, such as phytopathogenic oomycetes responsible for important crop diseases. Thanks to the negligible effects on human health and the environment, d-tagatose has been proposed as a sustainable product for crop protection. This review describes the current knowledge on modes of action of d-tagatose against phytopathogenic oomycetes and its potential uses in agriculture. d-tagatose can negatively affect the growth of phytopathogenic oomycetes by inhibiting key enzymes of sugar metabolism, such as β-glucosidase in Phytophthora infestans, and fructokinase and phosphomannose isomerase in Hyaloperonospora arabidopsidis. Moreover, d-tagatose affects sugar content, causes severe mitochondrial alterations, and inhibits respiration processes with the accumulation of reactive oxygen species in P. infestans, but not in P. cinnamomi. Differential effects of d-tagatose are associated with a global gene downregulation in P. infestans and with an efficient transcriptional reprogramming of multiple metabolic processes in P. cinnamomi. d-tagatose displays possible species-specific effects in Phytophthora spp. and nutritional properties on some plant-associated microorganisms. However, inhibitory effects are reversible and P. infestans growth can be restored in the absence of d-tagatose. Further functional studies are discussed in this review, in order to promote the use of d-tagatose for sustainable crop protectio

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

Time-resolved x-ray absorption spectroscopy with a water window high-harmonic source

Time-resolved X-ray absorption spectroscopy (TR-XAS) has so far practically been limited to large-scale facilities, to sub-picosecond temporal resolution and to the condensed phase. Here, we report the realization of TR-XAS with a temporal resolution in the low femtosecond range by developing a table-top high-harmonic source reaching up to 350 eV, thus partially covering the spectral region of 280 to 530 eV, where water is transmissive. We use this source to follow previously unexamined light-induced chemical reactions in the lowest electronic states of isolated CF4+ and SF6+ molecules in the gas phase. By probing element-specific core-to-valence transitions at the carbon K-edge or the sulfur L-edges, we characterize their reaction paths and observe the effect of symmetry breaking through the splitting of absorption bands and Rydberg-valence mixing induced by the geometry changes