RNA interference-based strategies to control Botrytis cinerea infection in cultivated strawberry.

KEY MESSAGE: Gene silencing of BcDCL genes improves gray mold disease control in the cultivated strawberry. Gene silencing technology offers new opportunities to develop new formulations or new pathogen-resistant plants for reducing impacts of agricultural systems. Recent studies offered the proof of concept that the symptoms of gray mold can be reduced by downregulating Dicer-like 1 (DCL1) and 2 (DCL2) genes of Botrytis cinerea. In this study, we demonstrate that both solutions based on dsRNA topical treatment and in planta expression targeting BcDCL1 and BcDCL2 genes can be used to control the strawberry gray mold, the most harmful disease for different fruit crops. 50, 70 and 100 ng µL-1 of naked BcDCL1/2 dsRNA, sprayed on plants of Fragaria x ananassa cultivar Romina in the greenhouse, displayed significant reduction of susceptibility, compared to the negative controls, but to a lesser extent than the chemical fungicide. Three independent lines of Romina cultivar were confirmed for their stable expression of the hairpin gene construct that targets the Bc-DCL1 and 2 sequences (hp-Bc-DCL1/2), and for the production of hp construct-derived siRNAs, by qRT-PCR and Northern blot analyses. In vitro and in vivo detached leaves, and fruits from the hp-Bc-DCL1/2 lines showed significantly enhanced tolerance to this fungal pathogen compared to the control. This decreased susceptibility was correlated to the reduced fungal biomass and the downregulation of the Bc-DCL1 and 2 genes in B. cinerea. These results confirm the potential of both RNAi-based products and plants for protecting the cultivated strawberry from B. cinerea infection, reducing the impact of chemical pesticides on the environment and the health of consumers.

First report of postharvest pear bitter rot caused by Colletotrichum acutatum in Italy.

The fungal genus Colletotrichum includes numerous important plant pathogenic species, some of which causes fruit bitter rot as well as leaf lesions (leaf black spot) on major crops, leading to yield losses (Fu et al. 2019; Talhinhas & Baroncelli, 2023). C. acutatum was reported associated with black spot on fallen, immature fruit of pear (Pyrus pyrifolia) in New Zealand (Damm et al. 2012); however, to our knowledge, this species has not been reported in Italy or nowhere else. In 2022, a significant increase of anthracnose symptoms was observed on pears in Emilia-Romagna region, Italy. Symptoms, such as round brown lesions of 1 to 4 cm, appeared on more than 50% of refrigerated stored fruit. These symptoms were undetectable at the end of September 2022 and appeared after a five-month period of storage (February 2023) at 4°C (e-Xtra 1A and B). Fungal isolates were obtained from symptomatic pears after surfaces sterilization with 96% ethanol by culturing necrotic tissue pieces on Potato Dextrose Agar at 25°C in the dark (e-Xtra 1C and D). Cultures were shades of coral color, from opalescent to sunkist coral, with slight aerial mycelium becoming grey and darker with age. When observed from the reverse side, the color was pink and, with age, became coral orange to dark amaranth. Conidia observed with a light microscope appeared hyaline and fusiform, 8 to 16 × 2.5 to 4 µm, with two pointed ends or one rounded end. (e-Xtra 1E) One reference isolate, named L51, was used for molecular characterization. Total genomic DNA was extracted, and the ITS region of rDNA amplified using the universal primers ITS1 and ITS4, then sequenced. The resulting sequences were 100% identical to those of C. acutatum (NR_144794.1: strain CBS 112996 ITS region; from TYPE material). Based on Damm et al. (2012), partial ACT, GAPDH, CHS and TUB2 gene sequences were also amplified and sequenced (GenBank Accession numbers: ITS: OR882016, ACT: OR882013, GAPDH: OR882011, CHS: OR882012, TUB2: OR882010), to characterize the isolates. Additionally, the multilocus phylogenetic analysis carried out with the obtained and reference sequences (Damm et al. 2012) revealed the species of analyzed isolates and confirmed the BLAST results, identifying the strain as C. acutatum (e-Xtra 1F). Koch's postulates were performed on 10 'Kaiser' pears. Surfaces sterilized fruits with 96% ethanol were subjected to wound inoculation with a conidial suspension (106 conidia ml-1) while 10 fruit were used as negative control and inoculated with sterile water. Following an incubation period of 8-14 days at 15-20°C, symptoms around the inoculation site resembled those initially observed, while the negative control showed no symptoms. Fungal colonies re-isolated from the lesions exhibited the same morphological characteristics as the original isolates. To our knowledge, this is the first report of pear bitter rot caused by C. acutatum in Italy and in Europe (Talhinhas & Baroncelli, 2023). Yet, bitter rot had not been recognized as a notable issue in pear cultivation. Nevertheless, given that pears rank as the 8th most cultivated fruit globally and economically very significant for the Emilia Romagna region in Italy the emergence of pear bitter rot caused by Colletotrichum species has the potential to evolve into a significant worldwide problem, warranting further investigation.

Functional characterization of MANNOSE-BINDING LECTIN 1, a G-type lectin gene family member, in response to fungal pathogens of strawberry.

The mannose-binding lectin gene MANNOSE-BINDING LECTIN 1 (MBL1) is a member of the G-type lectin family and is involved in defense in strawberry (Fragaria × ananassa). Genome-wide identification of the G-type lectin family was carried out in woodland strawberry, F. vesca, and 133 G-lectin genes were found. Their expression profiles were retrieved from available databases and indicated that many are actively expressed during plant development or interaction with pathogens. We selected MBL1 for further investigation and generated stable transgenic FaMBL1-overexpressing plants of F. ×ananassa to examine the role of this gene in defense. Plants were selected and evaluated for their contents of disease-related phytohormones and their reaction to biotic stresses, and this revealed that jasmonic acid decreased in the overexpressing lines compared with the wild-type (WT). Petioles of the overexpressing lines inoculated with Colletotrichum fioriniae had lower disease incidence than the WT, and leaves of these lines challenged by Botrytis cinerea showed significantly smaller lesion diameters than the WT and higher expression of CLASS II CHITINASE 2-1. Our results indicate that FaMBL1 plays important roles in strawberry response to fungal diseases caused by C. fioriniae and B. cinerea.

Complete Genome Sequence of the Plant-Pathogenic Fungus Colletotrichum lupini.

Colletotrichum is a fungal genus (Ascomycota, Sordariomycetes, Glomerellaceae) that includes many economically important plant pathogens that cause devastating diseases of a wide range of plants. In this work, using a combination of long- and short-read sequencing technologies, we sequenced the genome of Colletotrichum lupini RB221, isolated from white lupin (Lupinus albus) in France during a survey in 2014. The genome was assembled into 11 nuclear chromosomes and a mitochondrial genome with a total assembly size of 63.41 Mb and 36.55 kb, respectively. In total, 18,324 protein-encoding genes have been predicted, of which only 39 are specific to C. lupini. This resource will provide insight into pathogenicity factors and will help provide a better understanding of the evolution and genome structure of this important plant pathogen.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Aureobasidium pullulans volatile organic compounds as alternative postharvest method to control brown rot of stone fruits.

Volatile compounds produced by L1 and L8 strains were assayed against mycelia and conidia growth of Monilinia laxa, M. fructicola, M. polystroma, and M. fructigena of stone fruits. Results showed that volatile metabolites inhibited significantly pathogens growth, in particular M. fructigena mycelium growth (70% by L1 and 50% by L8) and M. fructicola conidia germination (85% by L1 and 70% by L8) compared to the control. Moreover, the antagonistic activity was enhanced by the addition of asparagine (120 mg L-1) in the culture media composition. Synthetic pure compounds were tested in vitro on pathogens mycelial and conidia growth and their EC50 values were estimated, confirming 2-phenethyl as the most active compound. For this reason 2-phenethyl and VOCs of both yeast strains were assayed in vivo on cherry, peach, and apricot fruits. Regarding peach fruit, both treatments, yeasts and pure compounds, displayed the best inhibiting action against all the pathogens especially against M. laxa (100% by L1, 84% by L8 and 2-phenethyl). ATR/IR spectroscopy analysis showed how VOCs produced by both strains increase the fruit waxes complexity reducing the pathogens attack so playing an essential role in the antagonistic activity of both yeast strains and on fruit structural composition.

Biotechnological Approaches: Gene Overexpression, Gene Silencing, and Genome Editing to Control Fungal and Oomycete Diseases in Grapevine.

Downy mildew, powdery mildew, and grey mold are some of the phytopathological diseases causing economic losses in agricultural crops, including grapevine, worldwide. In the current scenario of increasing global warming, in which the massive use of agrochemicals should be limited, the management of fungal disease has become a challenge. The knowledge acquired on candidate resistant (R) genes having an active role in plant defense mechanisms has allowed numerous breeding programs to integrate these traits into selected cultivars, even though with some limits in the conservation of the proper qualitative characteristics of the original clones. Given their gene-specific mode of action, biotechnological techniques come to the aid of breeders, allowing them to generate simple and fast modifications in the host, without introducing other undesired genes. The availability of efficient gene transfer procedures in grapevine genotypes provide valid tools that support the application of new breeding techniques (NBTs). The expertise built up over the years has allowed the optimization of these techniques to overexpress genes that directly or indirectly limit fungal and oomycetes pathogens growth or silence plant susceptibility genes. Furthermore, the downregulation of pathogen genes which act as virulence effectors by exploiting the RNA interference mechanism, represents another biotechnological tool that increases plant defense. In this review, we summarize the most recent biotechnological strategies optimized and applied on Vitis species, aimed at reducing their susceptibility to the most harmful fungal and oomycetes diseases. The best strategy for combating pathogenic organisms is to exploit a holistic approach that fully integrates all these available tools.

Different Antifungal Activity of Anabaena sp., Ecklonia sp., and Jania sp. against Botrytis cinerea.

Water extracts and polysaccharides from Anabaena sp., Ecklonia sp., and Jania sp. were tested for their activity against the fungal plant pathogen Botrytis cinerea. Water extracts at 2.5, 5.0, and 10.0 mg/mL inhibited B. cinerea growth in vitro. Antifungal activity of polysaccharides obtained by N-cetylpyridinium bromide precipitation in water extracts was evaluated in vitro and in vitro at 0.5, 2.0, and 3.5 mg/mL. These concentrations were tested against fungal colony growth, spore germination, colony forming units (CFUs), CFU growth, and on strawberry fruits against B. cinerea infection with pre- and post-harvest application. In in vitro experiments, polysaccharides from Anabaena sp. and from Ecklonia sp. inhibited B. cinerea colony growth, CFUs, and CFU growth, while those extracted from Jania sp. reduced only the pathogen spore germination. In in vitro experiments, all concentrations of polysaccharides from Anabaena sp., Ecklonia sp., and Jania sp. reduced both the strawberry fruits infected area and the pathogen sporulation in the pre-harvest treatment, suggesting that they might be good candidates as preventive products in crop protection.

Molecular analysis of the early interaction between the grapevine flower and Botrytis cinerea reveals that prompt activation of specific host pathways leads to fungus quiescence.

Grape quality and yield can be impaired by bunch rot, caused by the necrotrophic fungus Botrytis cinerea. Infection often occurs at flowering, and the pathogen stays quiescent until fruit maturity. Here, we report a molecular analysis of the early interaction between B. cinerea and Vitis vinifera flowers, using a controlled infection system, confocal microscopy and integrated transcriptomic and metabolic analysis of the host and the pathogen. Flowers from fruiting cuttings of the cultivar Pinot Noir were infected with green fluorescent protein (GFP)-labelled B. cinerea and studied at 24 and 96 hours post-inoculation (h.p.i.). We observed that penetration of the epidermis by B. cinerea coincided with increased expression of genes encoding cell-wall-degrading enzymes, phytotoxins and proteases. Grapevine responded with a rapid defence reaction involving 1193 genes associated with the accumulation of antimicrobial proteins, polyphenols, reactive oxygen species and cell wall reinforcement. At 96 h.p.i., the reaction appears largely diminished both in the host and in the pathogen. Our data indicate that the defence responses of the grapevine flower collectively are able to restrict invasive fungal growth into the underlying tissues, thereby forcing the fungus to enter quiescence until the conditions become more favourable to resume pathogenic development.

Unfoldome variation upon plant-pathogen interactions: strawberry infection by Colletotrichum acutatum.

Intrinsically disordered proteins (IDPs) are proteins that lack secondary and/or tertiary structure under physiological conditions. These proteins are very abundant in eukaryotic proteomes and play crucial roles in all molecular mechanisms underlying the response to environmental challenges. In plants, different IDPs involved in stress response have been identified and characterized. Nevertheless, a comprehensive evaluation of protein disorder in plant proteomes under abiotic or biotic stresses is not available so far. In the present work the transcriptome dataset of strawberry (Fragaria X ananassa) fruits interacting with the fungal pathogen Colletotrichum acutatum was actualized onto the woodland strawberry (Fragaria vesca) genome. The obtained cDNA sequences were translated into protein sequences, which were subsequently subjected to disorder analysis. The results, providing the first estimation of disorder abundance associated to plant infection, showed that the proteome activated in the strawberry red fruit during the active fungal propagation is remarkably depleted in disorder. On the other hand, in the resistant white fruit, no significant disorder reduction is observed in the proteins expressed in response to fungal infection. Four representative proteins, FvSMP, FvPRKRIP, FvPCD-4 and FvFAM32A-like, predicted as mainly disordered and never experimentally characterized before, were isolated, and the absence of structure was validated at the secondary and tertiary level using circular dichroism and differential scanning fluorimetry. Their quaternary structure was also established using light scattering. The results are discussed considering the role of protein disorder in plant defense.

Identification and characterization of the defensin-like gene family of grapevine.

Defensins are a class of small and diverse cysteine-rich proteins found in plants, insects, and vertebrates, which share a common tertiary structure and usually exert broad-spectrum antimicrobial activities. We used a bioinformatic approach to scan the Vitis vinifera genome and identified 79 defensin-like sequences (DEFL) corresponding to 46 genes and allelic variants, plus 33 pseudogenes and gene fragments. Expansion and diversification of grapevine DEFL has occurred after the split from the last common ancestor with the genera Medicago and Arabidopsis. Grapevine DEFL localization on the 'Pinot Noir' genome revealed the presence of several clusters likely evolved through local duplications. By sequencing reverse-transcription polymerase chain reaction products, we could demonstrate the expression of grapevine DEFL with no previously reported record of expression. Many of these genes are predominantly or exclusively expressed in tissues linked to plant reproduction, consistent with findings in other plant species, and some of them accumulated at fruit ripening. The transcripts of five DEFL were also significantly upregulated in tissues infected with Botrytis cinerea, a necrotrophic mold, suggesting a role of these genes in defense against this pathogen. Finally, three novel defensins were discovered among the identified DEFL. They inhibit B. cinerea conidia germination when expressed as recombinant proteins.

Development and validation of a real-time PCR assay for detection and quantification of Tuber magnatum in soil.

BACKGROUND: Tuber magnatum, the Italian white truffle, is the most sought-after edible ectomycorrhizal mushroom. Previous studies report the difficulties of detecting its mycorrhizas and the widespread presence of its mycelium in natural production areas, suggesting that the soil mycelium could be a good indicator to evaluate its presence in the soil. In this study a specific real-time PCR assay using TaqMan chemistry was developed to detect and quantify T. magnatum in soil. This technique was then applied to four natural T. magnatum truffières located in different regions of Italy to validate the method under different environmental conditions. RESULTS: The primer/probe sets for the detection and quantification of T. magnatum were selected from the ITS rDNA regions. Their specificity was tested in silico and using qualitative PCR on DNA extracted from 25 different fungal species. The T. magnatum DNA concentration was different in the four experimental truffières and higher in the productive plots. T. magnatum mycelium was however also detected in most of the non-productive plots. Ascoma production during the three years of the study was correlated with the concentration of T. magnatum DNA. CONCLUSIONS: Taken together, these results suggest that the specific real-time PCR assay perfected in this study could be an useful tool to evaluate the presence and dynamics of this precious truffle in natural and cultivated truffières.

Management of Post-Harvest Anthracnose: Current Approaches and Future Perspectives.

Anthracnose is a severe disease caused by Colletotrichum spp. on several crop species. Fungal infections can occur both in the field and at the post-harvest stage causing severe lesions on fruits and economic losses. Physical treatments and synthetic fungicides have traditionally been the preferred means to control anthracnose adverse effects; however, the urgent need to decrease the use of toxic chemicals led to the investigation of innovative and sustainable protection techniques. Evidence for the efficacy of biological agents and vegetal derivates has been reported; however, their introduction into actual crop protection strategies requires the solutions of several critical issues. Biotechnology-based approaches have also been explored, revealing the opportunity to develop innovative and safe methods for anthracnose management through genome editing and RNA interference technologies. Nevertheless, besides the number of advantages related to their use, e.g., the putative absence of adverse effects due to their high specificity, a number of aspects remain to be clarified to enable their introduction into Integrated Pest Management (IPM) protocols against Colletotrichum spp. disease.

Game-changing alternatives to conventional fungicides: small RNAs and short peptides.

Fungicide use is one of the core elements of intensive agriculture because it is necessary to fight pathogens that would otherwise cause large production losses. Oomycete and fungal pathogens are kept under control using several active compounds, some of which are predicted to be banned in the near future owing to serious concerns about their impact on the environment, non-targeted organisms, and human health. To avoid detrimental repercussions for food security, it is essential to develop new biomolecules that control existing and emerging pathogens but are innocuous to human health and the environment. This review presents and discusses the use of novel low-risk biological compounds based on small RNAs and short peptides that are attractive alternatives to current contentious fungicides.

Comparative transcriptomic provides novel insights into the soybean response to Colletotrichum truncatum infection.

Introduction: Soybean (Glycine max) is among the most important crops in the world, and its production can be threatened by biotic diseases, such as anthracnose. Soybean anthracnose is a seed-borne disease mainly caused by the hemibiotrophic fungus Colletotrichum truncatum. Typical symptoms are pre- and post-emergence damping off and necrotic lesions on cotyledons, petioles, leaves, and pods. Anthracnose symptoms can appear early in the field, causing major losses to soybean production. Material and Methods: In preliminary experiments, we observed that the same soybean cultivar can have a range of susceptibility towards different strains of C. truncatum, while the same C. truncatum strain can cause varying levels of disease severity in different soybean cultivars. To gain a better understanding of the molecular mechanisms regulating the early response of different soybean cultivars to different C. truncatum strains, we performed pathogenicity assays to select two soybean cultivars with significantly different susceptibility to two different C. truncatum strains and analyzed their transcriptome profiles at different time points of interaction (0, 12, 48, and 120 h post-inoculation, hpi). Results and Discussion: The pathogenicity assays showed that the soybean cultivar Gm1 is more resistant to C. truncatum strain 1080, and it is highly susceptible to strain 1059, while cultivar Gm2 shows the opposite behavior. However, if only trivial anthracnose symptoms appeared in the more resistant phenotype (MRP; Gm1-1080; Gm2-1059) upon 120 hpi, in the more susceptible phenotype (MSP; Gm-1059; Gm2- 1080) plants show mild symptoms already at 72 hpi, after which the disease evolved rapidly to severe necrosis and plant death. Interestingly, several genes related to different cellular responses of the plant immune system (pathogen recognition, signaling events, transcriptional reprogramming, and defense-related genes) were commonly modulated at the same time points only in both MRP. The list of differentially expressed genes (DEGs) specific to the more resistant combinations and related to different cellular responses of the plant immune system may shed light on the important host defense pathways against soybean anthracnose.

The activity of the plant mitochondrial inner membrane anion channel (PIMAC) of maize populations divergently selected for cold tolerance level is differentially dependent on the growth temperature of seedlings.

The activity of the plant inner membrane mitochondrial anion channel (PIMAC) is involved in metabolite shuttles and mitochondrial volume changes and could have a role in plant temperature tolerance. Our objectives were to investigate (i) the occurrence and (ii) the temperature dependence of anion fluxes through PIMAC in mitochondria isolated from seedlings of three maize populations differing in terms of cold tolerance; and (iii) the relationships between the PIMAC activity kinetics and the level of cold tolerance. Populations were the source population (C0) and two populations divergently selected for high (C4H) and low (C4L) cold tolerance. Such divergently selected populations are expected to share most of their genes, with the main exception of those genes controlling cold tolerance. Arrhenius plots of PIMAC chloride fluxes showed a linear temperature dependence when seedlings were grown at 25 or 14°C, whereas a non-linear temperature dependence was found when seedlings were grown at 5°C, with or without acclimation at 14°C. The activation energy and other thermodynamic parameters of PIMAC activity varied depending on temperature treatments during seedling growth. When seedlings were grown at 14 and 5°C with acclimation, PIMAC activity of the C4H population increased, while that of C4L declined, as compared with the activities of seedlings grown at 25°C. These symmetric responses indicate that PIMAC activity changes are associated with genetically determined differences in the cold tolerance level of the investigated populations. We conclude that anion fluxes by PIMAC depend upon changes on growth temperature and are differentially related to the tolerance level of the tested populations.

The activity of plant inner membrane anion channel (PIMAC) can be performed by a chloride channel (CLC) protein in mitochondria from seedlings of maize populations divergently selected for cold tolerance.

The proteins performing the activity of the inner membrane anion channel (IMAC) and its plant counterpart (PIMAC) are still unknown. Lurin et al. (Biochem J 348: 291-295, 2000) indicated that a chloride channel (CLC) protein corresponds to PIMAC activity in tobacco seedling mitochondria. In this study, we investigated: (i) the presence of a CLC protein in maize seedling mitochondria; (ii) the involvement of this protein in plant cold tolerance; and (iii) its possible role in PIMAC activity. We validated the presence of a CLC protein (ZmCLCc) in maize mitochondria by immunoassay using a polyclonal antibody against its C-terminus. The differential expression of the ZmCLCc protein in mitochondria was measured in seedlings of maize populations divergently selected for cold tolerance and grown at different temperatures. The ZmCLCc protein level was higher in cold stressed than in non-stressed growing conditions. Moreover, the ZmCLCc level showed a direct relationship with the cold sensitivity level of the populations under both growing conditions, suggesting that selection for cold tolerance induced a constitutive change of the ZmCLCc protein amount in mitochondria. The anti-ZmCLCc antibody inhibited (about 60%) the channel-mediated anion translocations by PIMAC, whereas the same antibody did not affect the free diffusion of potassium thiocyanide through the inner mitochondrial membrane. For this reason, we conclude that the mitochondrial ZmCLCc protein can perform the PIMAC activity in maize seedlings.

Does RNAi-Based Technology Fit within EU Sustainability Goals?

European Union (EU) and global sustainability policies emphasize the need to replace contentious pesticides with safe, efficient, and cost-effective alternatives to ensure sustainable food production. However, R&D for alternatives to contentious pesticides are lagging behind and need to be broadened. Here, we discuss how RNAi-based technology can contribute to pesticide risk reduction.

Double-Stranded RNA Targeting Dicer-Like Genes Compromises the Pathogenicity of Plasmopara viticola on Grapevine.

Downy mildew caused by Plasmopara viticola is one of the most devastating diseases of grapevine, attacking all green parts of the plant. The damage is severe when the infection at flowering stage is left uncontrolled. P. viticola management consumes a significant amount of classical pesticides applied in vineyards, requiring efficient and environmentally safe disease management options. Spray-induced gene silencing (SIGS), through the application of exogenous double-stranded RNA (dsRNA), has shown promising results for the management of diseases in crops. Here, we developed and tested the potential of dsRNA targeting P. viticola Dicer-like (DCL) genes for SIGS-based crop protection strategy. The exogenous application of PvDCL1/2 dsRNA, a chimera of PvDCL1 and PvDCL2, highly affected the virulence of P. viticola. The reduced expression level of PvDCL1 and PvDCL2 transcripts in infected leaves, treated with PvDCL1/2 dsRNA, was an indication of an active RNA interference mechanism inside the pathogen to compromise its virulence. Besides the protective property, the PvDCL1/2 dsRNA also exhibited a curative role by reducing the disease progress rate of already established infection. Our data provide a promising future for PvDCL1/2 dsRNA as a new generation of RNA-based resistant plants or RNA-based agrochemical for the management of downy mildew disease in grapevine.

RNA Interference Strategies for Future Management of Plant Pathogenic Fungi: Prospects and Challenges.

Plant pathogenic fungi are the largest group of disease-causing agents on crop plants and represent a persistent and significant threat to agriculture worldwide. Conventional approaches based on the use of pesticides raise social concern for the impact on the environment and human health and alternative control methods are urgently needed. The rapid improvement and extensive implementation of RNA interference (RNAi) technology for various model and non-model organisms has provided the initial framework to adapt this post-transcriptional gene silencing technology for the management of fungal pathogens. Recent studies showed that the exogenous application of double-stranded RNA (dsRNA) molecules on plants targeting fungal growth and virulence-related genes provided disease attenuation of pathogens like Botrytis cinerea, Sclerotinia sclerotiorum and Fusarium graminearum in different hosts. Such results highlight that the exogenous RNAi holds great potential for RNAi-mediated plant pathogenic fungal disease control. Production of dsRNA can be possible by using either in-vitro or in-vivo synthesis. In this review, we describe exogenous RNAi involved in plant pathogenic fungi and discuss dsRNA production, formulation, and RNAi delivery methods. Potential challenges that are faced while developing a RNAi strategy for fungal pathogens, such as off-target and epigenetic effects, with their possible solutions are also discussed.