Enhancement of herbicolin A production by integrated fermentation optimization and strain engineering in Pantoea agglomerans ZJU23.

BACKGROUND: The lipopeptide herbicolin A (HA) secreted by the biocontrol agent Pantoea agglomerans ZJU23 is a promising antifungal drug to combat fungal pathogens by targeting lipid rafts, both in agricultural and clinical settings. Improvement of HA production would be of great significance in promoting its commercialization. This study aims to enhance the HA production in ZJU23 by combining fermentation optimization and strain engineering. RESULTS: Based on the results in the single-factor experiments, corn steep liquor, temperature and initial pH were identified as the significant affecting factors by the Plackett-Burman design. The fermentation medium and conditions were further optimized using the Box-Behnken response surface method, and the HA production of the wild type strain ZJU23 was improved from ~ 87 mg/mL in King's B medium to ~ 211 mg/mL in HA induction (HAI) medium. A transposon library was constructed in ZJU23 to screen for mutants with higher HA production, and two transcriptional repressors for HA biosynthesis, LrhA and PurR, were identified. Disruption of the LrhA gene led to increased mRNA expression of HA biosynthetic genes, and subsequently improved about twofold HA production. Finally, the HA production reached ~ 471 mg/mL in the ΔLrhA mutant under optimized fermentation conditions, which is about 5.4 times higher than before (~ 87 mg/mL). The bacterial suspension of the ΔLrhA mutant fermented in HAI medium significantly enhanced its biocontrol efficacy against gray mold disease and Fusarium crown rot of wheat, showing equivalent control efficacies as the chemical fungicides used in this study. Furthermore, HA was effective against fungicide resistant Botrytis cinerea. Increased HA production substantially improved the control efficacy against gray mold disease caused by a pyrimethanil resistant strain. CONCLUSIONS: This study reveals that the transcriptional repressor LrhA negatively regulates HA biosynthesis and the defined HAI medium is suitable for HA production. These findings provide an extended basis for large-scale production of HA and promote biofungicide development based on ZJU23 and HA in the future.

New Insights on the Integrated Management of Plant Diseases by RNA Strategies: Mycoviruses and RNA Interference.

RNA-based strategies for plant disease management offer an attractive alternative to agrochemicals that negatively impact human and ecosystem health and lead to pathogen resistance. There has been recent interest in using mycoviruses for fungal disease control after it was discovered that some cause hypovirulence in fungal pathogens, which refers to a decline in the ability of a pathogen to cause disease. Cryphonectria parasitica, the causal agent of chestnut blight, has set an ideal model of management through the release of hypovirulent strains. However, mycovirus-based management of plant diseases is still restricted by limited approaches to search for viruses causing hypovirulence and the lack of protocols allowing effective and systemic virus infection in pathogens. RNA interference (RNAi), the eukaryotic cell system that recognizes RNA sequences and specifically degrades them, represents a promising. RNA-based disease management method. The natural occurrence of cross-kingdom RNAi provides a basis for host-induced gene silencing, while the ability of most pathogens to uptake exogenous small RNAs enables the use of spray-induced gene silencing techniques. This review describes the mechanisms behind and the potential of two RNA-based strategies, mycoviruses and RNAi, for plant disease management. Successful applications are discussed, as well as the research gaps and limitations that remain to be addressed.

N-acylhomoserine lactonase-based hybrid nanoflowers: a novel and practical strategy to control plant bacterial diseases.

BACKGROUND: The disease caused by plant pathogenic bacteria in the production, transportation, and storage of many crops has brought huge losses to agricultural production. N-acylhomoserine lactonases (AHLases) can quench quorum-sensing (QS) by hydrolyzing acylhomoserine lactones (AHLs), which makes them the promising candidates for controlling infections of QS-dependent pathogenic bacteria. Although many AHLases have been isolated and considered as a potentially effective preventive and therapeutic agents for bacterial diseases, the intrinsically poor ambient stability has seriously restricted its application. RESULTS: Herein, we showed that a spheroid enzyme-based hybrid nanoflower (EHNF), AhlX@Ni3(PO4)2, can be easily synthesized, and it exhibited 10 times AHL (3OC8-HSL) degradation activity than that with free AhlX (a thermostable AHL lactonase). In addition, it showed intriguing stability even at the working concentration, and retained ~ 100% activity after incubation at room temperature (25 °C) for 40 days and approximately 80% activity after incubation at 60 °C for 48 h. Furthermore, it exhibited better organic solvent tolerance and long-term stability in a complicated ecological environment than that of AhlX. To reduce the cost and streamline production processes, CSA@Ni3(PO4)2, which was assembled from the crude supernatants of AhlX and Ni3(PO4)2, was synthesized. Both AhlX@Ni3(PO4)2 and CSA@Ni3(PO4)2 efficiently attenuated pathogenic bacterial infection. CONCLUSIONS: In this study, we have developed N-acylhomoserine lactonase-based hybrid nanoflowers as a novel and efficient biocontrol reagent with significant control effect, outstanding environmental adaptability and tolerance. It was expected to overcome the bottlenecks of poor stability and limited environmental tolerance that have existed for over two decades and pioneered the practical application of EHNFs in the field of biological control.

Root-knot nematodes (Meloidogyne spp.) a threat to agriculture in Mexico: biology, current control strategies, and perspectives.

Root-knot nematodes (RKN) are sedentary parasites of the roots of plants and are considered some of the most damaging pests in agriculture. Since RKN target the root vascular system, they provoke host nutrient deprivation and defective water transport, causing above-ground symptoms of growth stunting, wilting, chlorosis, and reduced crop yields. In Mexico RKN infestations are primarily dealt with by treating with synthetic chemically based nematicides that are preferred by farmers over available bioproducts. However, due to environmental and human health concerns chemical control is increasingly restricted. Biological control of RKNs can help reduce the use of chemical nematicides as it is achieved with antagonistic organisms, mainly bacteria, fungi, other nematodes, or consortia of diverse microorganisms, which control nematodes directly by predation and parasitism at different stages: eggs, juveniles, or adults; or indirectly by the action of toxic diffusible inhibitory metabolites. The need to increase agricultural production and reduce negative environmental impact creates an opportunity for optimizing biological control agents to suppress nematode populations, but this endeavour remains challenging as researchers around the world try to understand diverse control mechanisms, nematode and microbe life cycles, ecology, metabolite production, predatory behaviours, molecular and biochemical interactions, in order to generate attractive products with the approval of local regulatory bodies. Here, we provide a brief review of the biology of the genus Meloidogyne, biological control strategies, and a comparison between chemical and bioproducts in the Mexican market, and guidelines emitted by national agencies to ensure safety and effectiveness of new developments.

Encapsulation of Plant Biocontrol Bacteria with Alginate as a Main Polymer Material.

One of the most favored trends in modern agriculture is biological control. However, many reports show that survival of biocontrol bacteria is poor in host plants. Providing biocontrol agents with protection by encapsulation within external coatings has therefore become a popular idea. Various techniques, including extrusion, spray drying, and emulsion, have been introduced for encapsulation of biocontrol bacteria. One commonly used biopolymer for this type of microencapsulation is alginate, a biopolymer extracted from seaweed. Recent progress has resulted in the production of alginate-based microcapsules that meet key bacterial encapsulation requirements, including biocompatibility, biodegradability, and support of long-term survival and function. However, more studies are needed regarding the effect of encapsulation on protective bacteria and their targeted release in organic crop production systems. Most importantly, the efficacy of alginate use for the encapsulation of biocontrol bacteria in pest and disease management requires further verification. Achieving a new formulation based on biodegradable polymers can have significant effects on increasing the quantity and quality of agricultural products.

Molecular Characterization and Taxonomic Assignment of Three Phage Isolates from a Collection Infecting Pseudomonas syringae pv. actinidiae and P. syringae pv. phaseolicola from Northern Italy.

Bacterial kiwifruit vine disease (Pseudomonas syringae pv. actinidiae, Psa) and halo blight of bean (P. syringae pv. phaseolicola, Pph) are routinely treated with copper, leading to environmental pollution and bacterial copper resistance. An alternative sustainable control method could be based on bacteriophages, as phage biocontrol offers high specificity and does not result in the spread of toxic residues into the environment or the food chain. In this research, specific phages suitable for phage-based biocontrol strategies effective against Psa and Pph were isolated and characterized. In total, sixteen lytic Pph phage isolates and seven lytic Psa phage isolates were isolated from soil in Piedmont and Veneto in northern Italy. Genome characterization of fifteen selected phages revealed that the isolated Pph phages were highly similar and could be considered as isolates of a novel species, whereas the isolated Psa phages grouped into four distinct clades, two of which represent putative novel species. No lysogeny-, virulence- or toxin-related genes were found in four phages, making them suitable for potential biocontrol purposes. A partial biological characterization including a host range analysis was performed on a representative subset of these isolates. This analysis was a prerequisite to assess their efficacy in greenhouse and in field trials, using different delivery strategies.

Transcriptome analysis reveals the molecular mechanisms of response to an emergent yellow-flower disease in green Chinese prickly ash (Zanthoxylum schinifolium).

Chinese prickly ash (Zanthoxylum) is extensively used as spice and traditional medicine in eastern Asian countries. Recently, an emergent yellow-flower disease (YFD) break out in green Chinese prickly ash (Zanthoxylum schinifolium, Qinghuajiao in Chinese) at Chongqing municipality, and then leads to a sharp reduction in the yield of Qinghuajiao, and thus results in great economic losses for farmers. To address the molecular response for the emergent YFD of Qinghuajiao, we analyzed the transcriptome of 12 samples including the leaves and inflorescences of asymptomatic and symptomatic plants from three different towns at Chongqing by high-throughput RNA-Seq technique. A total of 126,550 genes and 229,643 transcripts were obtained, and 21,054 unigenes were expressed in all 12 samples. There were 56 and 164 different expressed genes (DEGs) for the AL_vs_SL (asymptomatic leaf vs symptomatic leaf) and AF_vs_SF (asymptomatic flower vs symptomatic flower) groups, respectively. The results of KEGG analysis showed that the "phenylpropanoid biosynthesis" pathway that related to plant-pathogen interaction were found in AL_vs_SL and AF_vs_SF groups, and the "Plant-pathogen interaction" found in AF_vs_SF group, implying that this Qinghuajiao YFD might cause by plant pathogen. Interestingly, we detected 33 common unigenes for the 2 groups, and almost these unigenes were up-regulated in the symptomatic plants. Moreover, most of which were homologs to virus RNA, the components of viruses, implying that this YFD was related to virus. Our results provided a primary molecular basis for the prevention and treatment of YFD of Qinghuajiao trees.

Natural phytoalexin stilbene compound resveratrol and its derivatives as anti-tobacco mosaic virus and anti-phytopathogenic fungus agents.

Plant diseases caused by plant viruses and pathogens seriously affect crop yield and quality, and it is very difficult to control them. The discovery of new leads based on natural products is an important way to innovate pesticides. Based on the resveratrol is a kind of natural phytoalexin, but it cannot be used as candidate for the development of new drug due to its poor druggability. The phenolic hydroxyl groups in the resveratrol structure are easily destroyed by oxidation, in order to improve its stability, ester formation is the most commonly used modification method in drug design. Their structures were characterized by 1H NMR, 13C NMR and HRMS. The activity against tobacco mosaic virus (TMV) of these ester derivatives has been tested for the first time. The bioassay results showed part of the target compounds exhibited good to excellent in vivo activities against TMV. The optimum compounds III-2 (inhibitory rates of 50, 53, and 59% at 500 µg/mL for inactivation, curative, and protection activities in vivo, respectively), III-4 (inhibitory rates of 57, 59, and 51% at 500 µg/mL, respectively), and II-5 (inhibitory rates of 54, 52, and 51% at 500 µg/mL, respectively) displayed higher activity than commercial plant virucide ribavirin (inhibitory rates of 38, 37, and 40% at 500 µg/mL, respectively). Compounds I-9 and I-10 also showed excellent activities. The systematic study provides strong evidence that these simple resveratrol derivatives could become potential TMV inhibitors. The novel concise structure provides another new template for antiviral studies.

D-Amino Acid-Containing Lipopeptides Derived from the Lead Peptide BP100 with Activity against Plant Pathogens.

From a previous collection of lipopeptides derived from BP100, we selected 18 sequences in order to improve their biological profile. In particular, analogues containing a D-amino acid at position 4 were designed, prepared, and tested against plant pathogenic bacteria and fungi. The biological activity of these sequences was compared with that of the corresponding parent lipopeptides with all L-amino acids. In addition, the influence of the length of the hydrophobic chain on the biological activity was evaluated. Interestingly, the incorporation of a D-amino acid into lipopeptides bearing a butanoyl or a hexanoyl chain led to less hemolytic sequences and, in general, that were as active or more active than the corresponding all L-lipopeptides. The best lipopeptides were BP475 and BP485, both incorporating a D-Phe at position 4 and a butanoyl group, with MIC values between 0.8 and 6.2 µM, low hemolysis (0 and 24% at 250 µM, respectively), and low phytotoxicity. Characterization by NMR of the secondary structure of BP475 revealed that the D-Phe at position 4 disrupts the α-helix and that residues 6 to 10 are able to fold in an α-helix. This secondary structure would be responsible for the high antimicrobial activity and low hemolysis of this lipopeptide.

Bacteriophage therapy: recent developments and applications of a renaissant weapon.

Antimicrobial resistance is a global health problem and one of the leading concerns in healthcare sector. Bacteriophages are antibacterial agents ubiquitous in nature. With increase in antibiotic resistance, use of bacteriophages as therapeutics has become resurgent in recent times. This review focuses on the recent developments in phage therapy and its applications with respect to human infections, animal, food and environment. Moreover, use of phage proteins, bioengineered bacteriophages, and phage derived vaccines is also highlighted. Additionally, the limitations and challenges with regard to implementation of phage therapy, host safety and immune responses are also reviewed in this article.

Design, Synthesis and Antibacterial Activity of Novel Pyrimidine-Containing 4H-Chromen-4-One Derivatives*.

A series of pyrimidine-containing 4H-chromen-4-one derivatives were designed and synthesized by combining bioactive substructures. Preliminary biological activity results showed that most of the compounds displayed significant inhibitory activities in vitro against Xanthomonas axonopodis pv. Citri (X. axonopodis), Xanthomonas oryzae pv. oryzae (X. oryzae) and Ralstonia solanacearum (R. solanacearum). In particular, compound 2-[(3-{[5,7-dimethoxy-4-oxo-2-(3,4,5-trimethoxyphenyl)-4H-1-benzopyran-3-yl]oxy}propyl)sulfanyl]-4-(4-methylphenyl)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile (4c) demonstrated a good inhibitory effect against X. axonopodis and X. oryzae, with the half-maximal effective concentration (EC50 ) values of 15.5 and 14.9 µg/mL, respectively, and compound 2-[(3-{[5,7-Dimethoxy-4-oxo-2-(3,4,5-trimethoxyphenyl)-4H-1-benzopyran-3-yl]oxy}propyl)sulfanyl]-4-(3-fluorophenyl)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile (4h) showed the best antibacterial activity against R. solanacearum with an EC50 value of 14.7 µg/mL. These results were better than commercial reagents bismerthiazol (BT, 51.7, 70.1 and 52.7 µg/mL, respectively) and thiodiazole copper (TC, 77.9, 95.8 and 72.1 µg/mL, respectively). In vivo antibacterial activity results indicated that compound 4c displayed better curative (42.4 %) and protective (49.2 %) activities for rice bacterial leaf blight than BT (35.2, 39.1 %) and TC (30.8, 27.3 %). The mechanism of compound 4c against X. oryzae was analyzed through scanning electron microscopy (SEM). These results indicated that pyrimidine-containing 4H-chromen-4-one derivatives have important value in the research of new agrochemicals.

Soil microbiome manipulation triggers direct and possible indirect suppression against Ralstonia solanacearum and Fusarium oxysporum.

Soil microbiome manipulation can potentially reduce the use of pesticides by improving the ability of soils to resist or recover from pathogen infestation, thus generating natural suppressiveness. We simulated disturbance through soil fumigation and investigated how the subsequent application of bio-organic and organic amendments reshapes the taxonomic and functional potential of the soil microbiome to suppress the pathogens Ralstonia solanacearum and Fusarium oxysporum in tomato monocultures. The use of organic amendment alone generated smaller shifts in bacterial and fungal community composition and no suppressiveness. Fumigation directly decreased F. oxysporum and induced drastic changes in the soil microbiome. This was further converted from a disease conducive to a suppressive soil microbiome due to the application of organic amendment, which affected the way the bacterial and fungal communities were reassembled. These direct and possibly indirect effects resulted in a highly efficient disease control rate, providing a promising strategy for the control of the diseases caused by multiple pathogens.

Isolation and Characterization of a Novel Jumbo Phage from Leaf Litter Compost and Its Suppressive Effect on Rice Seedling Rot Diseases.

Jumbo phages have DNA genomes larger than 200 kbp in large virions composed of an icosahedral head, tail, and other adsorption structures, and they are known to be abundant biological substances in nature. In this study, phages in leaf litter compost were screened for their potential to suppress rice seedling rot disease caused by the bacterium Burkholderia glumae, and a novel phage was identified in a filtrate-enriched suspension of leaf litter compost. The phage particles consisted of a rigid tailed icosahedral head and contained a DNA genome of 227,105 bp. The phage could lyse five strains of B. glumae and six strains of Burkholderia plantarii. The phage was named jumbo Burkholderia phage FLC6. Proteomic tree analysis revealed that phage FLC6 belongs to the same clade as two jumbo Ralstonia phages, namely RSF1 and RSL2, which are members of the genus Chiangmaivirus (family: Myoviridae; order: Caudovirales). Interestingly, FLC6 could also lyse two strains of Ralstonia pseudosolanacearum, the causal agent of bacterial wilt, suggesting that FLC6 has a broad host range that may make it especially advantageous as a bio-control agent for several bacterial diseases in economically important crops. The novel jumbo phage FLC6 may enable leaf litter compost to suppress several bacterial diseases and may itself be useful for controlling plant diseases in crop cultivation.

Selection and characterization of two Bacillus thuringiensis strains showing nematicidal activity against Caenorhabditis elegans and Meloidogyne incognita.

Bacillus thuringiensis has been widely used as a biological control agent against insect pests. Additionally, nematicidal strains have been under investigation. In this report, 310 native strains of B. thuringiensis against Caenorhabditis elegans were tested. Only the LBIT-596 and LBIT-107 strains showed significant mortality. LC50s of spore-crystal complexes were estimated at 37.18 and 31.89 µg/mL for LBIT-596 and LBIT-107 strains, respectively, while LC50s of partially purified crystals was estimated at 23.76 and 20.25 µg/mL for LBIT-596 and LBIT-107, respectively. The flagellin gene sequence and plasmid patterns indicated that LBIT-596 and LBIT-107 are not related to each other. Sequences from internal regions of a cry5B and a cyt1A genes were found in the LBIT-596 strain, while a cry21A, a cry14A and a cyt1A genes were found in the LBIT-107 strain. Genome sequence of the LBIT-107 strain showed new cry genes, along with other virulence factors, hence, total nematicidal activity of the LBIT-107 strain may be the result of a multifactorial effect. The highlight of this contribution is that translocation of spore-crystal suspensions of LBIT-107 into tomato plants inoculated at their rhizosphere decreased up to 90% the number of galls of Meloidogyne incognita, perhaps the most important nematode pest in the world.

Order of microbial succession affects rhizobia-mediated biocontrol efforts against Phytophthora root rot.

The management of soilborne root diseases in pulse crops is challenged by a limited range of resistance sources and often a complete absence of in-crop management options. Therefore, alternative management strategies need to be developed. We evaluated disease limiting interactions between the rhizobia species Mesorhizobium ciceri, and the oomycete pathogen Phytophthora medicaginis, which causes Phytophthora root rot (PRR) of chickpea (Cicer arietinum). For the PRR susceptible var. Sonali plants, post-pathogen M. ciceri inoculation significantly improved probability of plant survival when compared to P. medicaginis infected plants only pre-inoculated with M. ciceri (75 % versus 35 %, respectively). Potential mechanisms for these effects were investigated: rhizobia inoculation benefits to plant nodulation were not demonstrated, but the highest nodule N-fixation activity of P. medicaginis inoculated plants occurred for the post-pathogen M. ciceri treatment; rhizobia inoculation treatment did not reduce lesion development but certain combinations of microbial inoculation led to significant reduction in root growth. Microcosm studies, however, showed that the presence of M. ciceri reduced growth of P. medicaginis isolates. Putative chickpea disease resistance gene expression was evaluated using qPCR in var. Sonali roots. When var. Sonali plants were treated with M. ciceri post-P. medicaginis inoculation, the gene regulation in the plant host became more similar to PRR moderately resistant var. PBA HatTrick. These results suggest that M. ciceri application post P. medicaginis inoculation may improve plant survival by inducing defense responses similar to a PRR moderately resistant chickpea variety. Altogether, these results indicate that order of microbial succession can significantly affect PRR plant survial in susceptible chickpea under controlled conditions and improved plant survival effects are due to a number of different mechanisms including improved host nutrition, through direct inhibiton of pathogen growth, as well as host defense priming.

Review on Structures of Pesticide Targets.

Molecular targets play important roles in agrochemical discovery. Numerous pesticides target the key proteins in pathogens, insect, or plants. Investigating ligand-binding pockets and/or active sites in the proteins' structures is usually the first step in designing new green pesticides. Thus, molecular target structures are extremely important for the discovery and development of such pesticides. In this manuscript, we present a review of the molecular target structures, including those of antiviral, fungicidal, bactericidal, insecticidal, herbicidal, and plant growth-regulator targets, currently used in agrochemical research. The data will be helpful in pesticide design and the discovery of new green pesticides.

NoPv1: a synthetic antimicrobial peptide aptamer targeting the causal agents of grapevine downy mildew and potato late blight.

Grapevine (Vitis vinifera L.) is a crop of major economic importance. However, grapevine yield is guaranteed by the massive use of pesticides to counteract pathogen infections. Under temperate-humid climate conditions, downy mildew is a primary threat for viticulture. Downy mildew is caused by the biotrophic oomycete Plasmopara viticola Berl. & de Toni, which can attack grapevine green tissues. In lack of treatments and with favourable weather conditions, downy mildew can devastate up to 75% of grape cultivation in one season and weaken newly born shoots, causing serious economic losses. Nevertheless, the repeated and massive use of some fungicides can lead to environmental pollution, negative impact on non-targeted organisms, development of resistance, residual toxicity and can foster human health concerns. In this manuscript, we provide an innovative approach to obtain specific pathogen protection for plants. By using the yeast two-hybrid approach and the P. viticola cellulose synthase 2 (PvCesA2), as target enzyme, we screened a combinatorial 8 amino acid peptide library with the aim to identify interacting peptides, potentially able to inhibit PvCesa2. Here, we demonstrate that the NoPv1 peptide aptamer prevents P. viticola germ tube formation and grapevine leaf infection without affecting the growth of non-target organisms and without being toxic for human cells. Furthermore, NoPv1 is also able to counteract Phytophthora infestans growth, the causal agent of late blight in potato and tomato, possibly as a consequence of the high amino acid sequence similarity between P. viticola and P. infestans cellulose synthase enzymes.

Synthesis and antifungal activities of hydrophilic cationic polymers against Rhizoctonia solani.

A series of linear hydrophilic cationic polymers with different charge density and molecular weights were synthesized by one-step polymerization process. The effect of the hydrophobicity and molecular weights on the antifungal activity against Rhizoctonia solani (R. solani) and Fusarium oxysporum f. sp. cubense race 4 (Foc4) was assessed. The biotoxicity of the cationic polymers were evaluated based on their median lethal concentration (LC50) for zebrafish and silkworm and median lethal dose (LD50) for Kunming mice. The results indicated that the balance between antifungal activity and biotoxicity could be well tuned by controlling the hydrophobic-hydrophilic balance. The minimum inhibitory concentration (MIC) of PEPB10 and PEPB25 against R. solani were 160 µg/mL and 80 µg/mL, respectively. And the LD50 for Kunming mice of PEPB10 and PEPB25 were more than 5000 mg/kg, which mean that PEPB10 and PEPB25 with high hydrophilicity show low toxicity and better selectivity for R. solani. The cationic polymers can kill the R. solani by damaging their membranes and exchanging the Ca2+ or/and Mg2+ cations of their membranes or cell wall. These results help to understand the antifungal mechanism of low-toxic polymeric quaternary ammonium salts and highlight their potential application as highly selective fungicidal agents for controlling plant diseases.

Synthesizing Fluorescently Labeled dsRNAs and sRNAs to Visualize Fungal RNA Uptake.

Fungal pathogens are responsible for severe crop losses worldwide. Defending crops against fungal disease is critical for global food security; however, most current disease management approaches rely on chemical fungicides that can leave dangerous residues in the environment. RNA interference (RNAi) is an important process through which RNA molecules target and silence complementary genes, regulating gene expression during both transcription and translation. Recently, it has been discovered that some species of fungi can efficiently take up RNAs originating from their host plant and the environment. If these RNAs are complementary to fungal genes, this can lead to the targeting and silencing of fungal genes, termed "cross-kingdom RNAi," if the RNA originated from a plant host, or "environmental RNAi," if the RNA originated from the environment. These discoveries have inspired the development of spray-induced gene silencing (SIGS), an innovative crop protection strategy involving the foliar application of RNAs which target and silence fungal virulence genes for plant protection against fungal pathogens. The effectiveness of SIGS is largely dependent on the ability of fungi to take up environmental RNAs. Here, we describe the protocols used to label and visualize RNAs which are taken up by Botrytis cinerea. This protocol could easily be adapted for use across various fungal species. Determining the efficiency of RNA uptake by a specific fungal species is a critical first step to determining if SIGS approaches could be an effective control strategy for that fungus.

Elicitins as molecular weapons against pathogens: consolidated biotechnological strategy for enhancing plant growth.

To fight against pathogens, defense systems in plants mainly depend upon preformed as well as induced responses. Pathogen detection activates induced responses and signals are transmitted for coordinated cellular events in order to restrict infection and spread. In spite of significant developments in manipulating genes, transcription factors and proteins for their involvement in immunity, absolute tolerance/resistance to pathogens has not been seen in plants/crops. Defense responses, among diverse plant types, to different pathogens involve modifications at the physio-biochemical and molecular levels. Secreted by oomycetes, elicitins are small, highly conserved and sterol-binding extracellular proteins with PAMP (pathogen associated molecular patterns) functions and are capable of eliciting plant defense reactions. Belonging to multigene families in oomycetes, elicitins are different from other plant proteins and show a different affinity for binding sterols and other lipids. These function for sterols binding to catalyze their inter-membrane and intra- as well as inter-micelle transport. Importantly, elicitins protect plants by inducing HR (hypersensitive response) and systemic acquired resistance. Despite immense metabolic significance and the involvement in defense activities, elicitins have not yet been fully studied and many questions regarding their functional activities remain to be explained. In order to address multiple questions associated with the role of elicitins, we have reviewed the understanding and topical advancements in plant defense mechanisms with a particular interest in elicitin-based defense actions and metabolic activities. This article offers potential attributes of elicitins as the biological control of plant diseases and can be considered as a baseline toward a more profound understanding of elicitins.