Challenges and Opportunities Arising from Host-Botrytis cinerea Interactions to Outline Novel and Sustainable Control Strategies: The Key Role of RNA Interference.

The necrotrophic plant pathogenic fungus Botrytis cinerea (Pers., 1794), the causative agent of gray mold disease, causes significant losses in agricultural production. Control of this fungal pathogen is quite difficult due to its wide host range and environmental persistence. Currently, the management of the disease is still mainly based on chemicals, which can have harmful effects not only on the environment and on human health but also because they favor the development of strains resistant to fungicides. The flexibility and plasticity of B. cinerea in challenging plant defense mechanisms and its ability to evolve strategies to escape chemicals require the development of new control strategies for successful disease management. In this review, some aspects of the host-pathogen interactions from which novel and sustainable control strategies could be developed (e.g., signaling pathways, molecules involved in plant immune mechanisms, hormones, post-transcriptional gene silencing) were analyzed. New biotechnological tools based on the use of RNA interference (RNAi) are emerging in the crop protection scenario as versatile, sustainable, effective, and environmentally friendly alternatives to the use of chemicals. RNAi-based fungicides are expected to be approved soon, although they will face several challenges before reaching the market.

Knockdown of Bmp1 and Pls1 Virulence Genes by Exogenous Application of RNAi-Inducing dsRNA in Botrytis cinerea.

Botrytis cinerea is a pathogen of wide agronomic and scientific importance partly due to its tendency to develop fungicide resistance. Recently, there has been great interest in the use of RNA interference as a control strategy against B. cinerea. In order to reduce the possible effects on non-target species, the sequence-dependent nature of RNAi can be used as an advantage to customize the design of dsRNA molecules. We selected two genes related to virulence: BcBmp1 (a MAP kinase essential for fungal pathogenesis) and BcPls1 (a tetraspanin related to appressorium penetration). After performing a prediction analysis of small interfering RNAs, dsRNAs of 344 (BcBmp1) and 413 (BcPls1) nucleotides were synthesized in vitro. We tested the effect of topical applications of dsRNAs, both in vitro by a fungal growth assay in microtiter plates and in vivo on artificially inoculated detached lettuce leaves. In both cases, topical applications of dsRNA led to gene knockdown with a delay in conidial germination for BcBmp1, an evident growth retardation for BcPls1, and a strong reduction in necrotic lesions on lettuce leaves for both genes. Furthermore, a strongly reduced expression of the BcBmp1 and BcPls1 genes was observed in both in vitro and in vivo experiments, suggesting that these genes could be promising targets for the development of RNAi-based fungicides against B. cinerea.

Innovative Approaches for Crop Improvement and Sustainable Management of Plant Disease in the Post-Genomic Era.

Safeguarding food supply in a world environment subject to sudden climate change, reducing the use of anthropogenic sources of pollution as much as possible, and using crops that must necessarily be increasingly resilient to biotic and abiotic stresses is a mandatory and ambitious necessity for the foreseeable future [...].

Silencing of the Slt2-Type MAP Kinase Bmp3 in Botrytis cinerea by Application of Exogenous dsRNA Affects Fungal Growth and Virulence on Lactuca sativa.

Botrytis cinerea can attack over 500 genera of vascular plants and is considered the second phytopathogen in the 'top ten' for its economic importance. Traditional fungicides can be ineffective and with increasing fungicide resistance, new sustainable technologies are required. Lately, RNA interference-based fungicides are emerging for their potential uses in crop protection. Therefore, we assessed the potential of this innovative approach targeting the MAP kinase Bmp3 in B. cinerea, a gene involved in saprophytic growth, response to low osmolarity, conidiation, surface sensing, host penetration and lesion formation. After performing a prediction analysis of small interfering RNAs, a 427 nucleotides long dsRNA was selected as construct. We tested the effect of topical applications of dsRNA construct both in vitro by a fungal growth assay in microtiter plates and in vivo on detached lettuce leaves artificially inoculated. In both cases, topical applications of dsRNA led to gene knockdown with a delay in conidial germination, an evident growth retardation and a strong reduction of necrotic lesions on leaves. These results correlated with a strongly reduced expression of Bmp3 gene. In accordance to these findings, the Bmp3 gene could be a promising target for the development of an RNAi-based fungicide against B. cinerea.

Molecular Detection of the Seed-Borne Pathogen Colletotrichum lupini Targeting the Hyper-Variable IGS Region of the Ribosomal Cluster.

Lupins anthracnose is a destructive seed and airborne disease caused by Colletotrichum lupini, affecting stems and pods. Primary seed infections as low as 0.01-0.1% can cause very severe yield losses. One of the most effective management strategies is the development of a robust and sensitive seed detection assay to screen seed lots before planting. PCR-based detection systems exhibit higher levels of sensitivity than conventional techniques, but when applied to seed tests they require the extraction of PCR-quality DNA from target organisms in backgrounds of saprophytic organisms and inhibitory seed-derived compounds. To overcome these limitations, a new detection protocol for C. lupini based on a biological enrichment step followed by a PCR assay was developed. Several enrichment protocols were compared with Yeast Malt Broth amended with ampicillin, streptomycin, and lactic acid were the most efficient. A species-specific C. lupini primer pair was developed based on rDNA IGS sequences. The specificity was evaluated against 17 strains of C. lupini, 23 different Colletotrichum species, and 21 different organisms isolated from seeds of Lupinus albus cv. Multitalia, L. luteus cv. Mister, and L. angustifolius cv. Tango. The protocol described here enabled the detection of C. lupini in samples artificially infected with less than 1/10,000 infected seed.

Development of a rapid PCR-Nucleic Acid Lateral Flow Immunoassay (PCR-NALFIA) based on rDNA IGS sequence analysis for the detection of Macrophomina phaseolina in soil.

The 'Nucleic Acid Lateral Flow Immunoassay' (NALFIA) using a generic 'Lateral Flow Device' (LFD), combined with PCR employing labelled primers (PCR-NALFIA), enables to circumvent the use of electrophoresis, making the diagnostic procedure more rapid and easier. If the specific amplicon is present in the sample, a coloured band, with an intensity proportional to the amplicon concentration, will develop on the LFD strip in addition to the control band. Species-specific primers for M. phaseolina based on the rDNA intergenic spacer (IGS) were developed and their specificity was checked and confirmed using 20 isolates of M. phaseolina and other 16 non-target fungi. A DNA extraction protocol based on a bead-beating technique using silica beads, skimmed milk and PVP was also developed. The M. phaseolina specific primers MP102F/MP102R, 5' labelled with biotin and FITC respectively, were used in the PCR-NALFIA assay to identify the pathogen starting from mycelium or microsclerotia. Microsclerotia of M. phaseolina (1, 10, 100 and 200) were manipulated under a stereomicroscope and their DNA was extracted using microsclerotia alone or mixed with different types of soil. The resulting DNA, used for the PCR-NALFIA assay, provided positive results for all the samples tested. A semi-quantitative grey-scale reference card based on the PCR-NALFIA assay using intervals corresponding to microsclerotia soil number was developed. For this purpose, the normalized pixel grey volumes obtained after a densitometric analysis of the test line intensity generated by the LFD dipsticks were used.

Effects of ozone exposure or fungal pathogen on white lupin leaves as determined by imaging of chlorophyll a fluorescence.

Chlorophyll fluorescence has been used routinely to investigate photosynthetic activity in plants subjected to both biotic and abiotic stresses. The aim of this work was to compare the perturbations in photosynthesis induced by ozone and by a pathogen. By using a conventional fluorometer a similar response pattern was observed in inoculated and O(3)-fumigated leaves. The application of chlorophyll fluorescence imaging provided further detailed information on the spatial-temporal heterogeneity of the response of white lupin leaves to fungal pathogen or to ozone fumigation. In particular, 48 h after artificial inoculation with the necrotrophic fungal pathogen Pleiochaeta setosa, the leaves showed a remarkable alteration in PSII operating efficiency (Phi(PSII)), which affected the whole surface. Afterwards, the infection site was surrounded by a ring of increased photosynthetic activity. The response of ozonated leaves was quite different. The reduction in Phi(PSII) was already evident 24h after fumigation; moreover, a distinct heterogeneity of the fluorescence yield was observed and the major veins displayed a lowered Phi(PSII).

Intraspecific diversity within Diaporthe helianthi: evidence from rDNA intergenic spacer (IGS) sequence analysis.

Diaporthe helianthi is the causal agent of sunflower stem canker, a serious pathogen of sunflower in Europe but recorded sporadically in Italy. The genetic diversity of D. helianthi isolates from different geographic origins (Argentina, France, Italy, Yugoslavia, Romania) was investigated using IGS sequences. A 400 bp fragment of the portion of the IGS region flanking the 5' end of the 18S gene was amplified from each isolate. The aligned nucleotide sequences showed intraspecific sequence homology from 99-100% among French/Yugoslavian isolates to 95-100% among Italian isolates. French/Yugoslavian isolates shared 90-92% sequence homology with Italian isolates. The phylogenetic tree obtained from the aligned data revealed three separate groups. Group 1 included all isolates from France and former Yugoslavia and one isolate from Argentina; Group 2 included all Italian isolates and one isolate from Argentina. The most distantly related isolate was that from Romania (Group 3). The average genetic distances among isolates within Group 1 and within Group 2 were 0.22 and 3.29 respectively. The analysis showed that all isolates originating from countries where severe outbreaks of the disease are reported annually (France and former Yugoslavia) form a well defined taxon characterized by relatively low variability. This group is distinct from the group formed by isolates originating from Italy, whose variability is relatively much higher. Results obtained revealed a marked differentiation among pathogen isolates, and members of Group 1 seem not yet to have spread into Italian sunflower-growing areas.