Row1, a member of a new family of conserved fungal proteins involved in infection, is required for appressoria functionality in Ustilago maydis.

The appressorium of phytopathogenic fungi is a specific structure with a crucial role in plant cuticle penetration. Pathogens with melanized appressoria break the cuticle through cell wall melanization and intracellular turgor pressure. However, in fungi with nonmelanized appressorium, the mechanisms governing cuticle penetration are poorly understood. Here we characterize Row1, a previously uncharacterized appressoria-specific protein of Ustilago maydis that localizes to membrane and secretory vesicles. Deletion of row1 decreases appressoria formation and plant penetration, thereby reducing virulence. Specifically, the Δrow1 mutant has a thicker cell wall that is more resistant to glucanase degradation. We also observed that the Δrow1 mutant has secretion defects. We show that Row1 is functionally conserved at least among Ustilaginaceae and belongs to the Row family, which consists of five other proteins that are highly conserved among Basidiomycota fungi and are involved in U. maydis virulence. We observed similarities in localization between Row1 and Row2, which is also involved in cell wall remodelling and secretion, suggesting similar molecular functions for members of this protein family. Our data suggest that Row1 could modify the chitin-glucan matrix of the fungal cell wall and may be involved in unconventional protein secretion, thereby promoting both appressoria maturation and penetration.

Surface wax in the ancestral grapevine Vitis sylvestris correlate with partial resistance to Powdery Mildew.

BACKGROUND: Powdery Mildew of Grapevine belongs to the major diseases in viticulture and requires intensive use of fungicides. Genetic introgression of resistance factors from wild grapes from North America and, recently, China, has been successful, but wine made from those varieties is still confronted with low consumer acceptance, due to differences in taste. RESULTS: The current work explores the potential of Vitis vinifera sylvestris, the wild ancestor of domesticated Grapevine, with respect to containing Erysiphe necator, the causative agent of Powdery Mildew. Making use of a germplasm collection comprising the entire genetic variability remaining in Germany, we show that there is considerable genetic variation in the formation of leaf surface waxes exceeding wax formation in commercial varieties. CONCLUSIONS: High wax formation correlates with reduced susceptibility to controlled infection with E. necator linked with perturbations of appressoria formation. We propose V. vinifera sylvestris as novel source for resistance breeding since it is genetically much closer to domesticated grapevine than the hitherto used sources from beyond the species barrier.

Septum-associated microtubule organizing centers within conidia support infectious development by the blast fungus Magnaporthe oryzae.

Cytoplasmic microtubule arrays play important and diverse roles within fungal cells, including serving as molecular highways for motor-driven organelle motility. While the dynamic plus ends of cytoplasmic microtubules are free to explore the cytoplasm through their stochastic growth and shrinkage, their minus ends are nucleated at discrete organizing centers, composed of large multi-subunit protein complexes. The location and composition of these microtubule organizing centers varies depending on genus, cell type, and in some instances cell-cycle stage. Despite their obvious importance, our understanding of the nature, diversity, and regulation of microtubule organizing centers in fungi remains incomplete. Here, using three-color fluorescence microscopy based live-cell imaging, we investigate the organization and dynamic behavior of the microtubule cytoskeleton within infection-related cell types of the filamentous fungus,Magnaporthe oryzae, a highly destructive pathogen of rice and wheat. We provide data to support the idea that cytoplasmic microtubules are nucleated at septa, rather than at nuclear spindle pole bodies, within the three-celled blast conidium, and provide new insight into remodeling of the microtubule cytoskeleton during nuclear division and inheritance. Lastly, we provide a more complete picture of the architecture and subcellular organization of the prototypical blast appressorium, a specialized pressure-generating cell type used to invade host tissue. Taken together, our study provides new insight into microtubule nucleation, organization, and dynamics in specialized and differentiated fungal cell types.

Gcc1 homologs regulate growth, oxidative stress, conidiation and appressorium formation in Colletotrichum siamense and Colletotrichum graminicola.

The Zn2Cys6 transcription factor is a fungal-specific zinc finger protein, which plays an important role in regulating growth, development and pathogenicity of pathogenic fungi. In this study, we characterized two Zn2Cys6 transcription factors, CsGcc1 and CgrGcc1 in Colletotrichum siamense and C. graminicola, respectively, which are homologous to Gcc1 in Magnaporthe oryzae. Both CsGcc1 and CgrGcc1 contain a typical GAL4 DNA-binding domain. Deletion of CsGCC1 or CgrGCC1 decreased the growth rate and lowered the tolerance to H2O2. In addition, disrupting CsGCC1 reduced conidial yield and lowered the germination rate and appressorium formation rate of C. siamense. Cellophane assays showed that deletion of CsGCC1 also weakened the penetration ability of appressoria. In C. graminicola, CgrGcc1 did not affect the production and germination of oval conidia, but its deletion significantly decreased the yield of the falcate conidium, and led to abnormal appressorium formation. In terms of pathogenicity, CsGcc1 slightly reduced the virulence of C. siamense, while deleting CgrGcc1 did not affect virulence of C. graminicola. In conclusion, the Zn2Cys6 transcription factors CsGcc1 and CgrGcc1 are involved in the regulation of vegetative growth, oxidative stress, conidial/falcate conidial production and appressorium formation in C. siamense and C. graminicola.

The Role of Fatty Acids from Plant Surfaces in the Infectivity of Colletotrichum fioriniae.

Aqueous extracts derived from flowers stimulate germination, secondary conidiation, and appressorial formation of various latent fruit rotting fungi. Even raindrops passing over flowers accumulate sufficient activity to influence the infectivity of fruit rotting fungi. Using a spore germination bioassay, high levels of bioactivity were found in chloroform extracts from plant tissues, implicating the nonpolar components of the cuticle. The fatty acid (FA) and fatty acid methyl ester (FAME) composition (C9-C20) of blueberry and cranberry tissues as well as aqueous flower extracts were characterized using a gas chromatography-mass spectrometry (GC-MS) method. The FAs and FAMEs found in the plant extracts were then tested for bioactivity using a spore germination bioassay. The C16:0 and C18:2 FAs and FAMEs, as well as the C18:0 FAME and the C20:0 FA, all stimulated appressorial formation while the C10:0 FA stimulated secondary conidiation. The C10:0 and C16:0 FAs were the only two bioactive components also identified from the aqueous floral extracts of both blueberry and cranberry and are therefore considered as contributors to the bioactivity observed in these extracts. The aqueous extracts from surfaces other than flowers showed little or no activity, and it is speculated that the movement of FAs may be related to the level of polymerization and cutin polyester development in flowers versus other plant organs. This study highlights the importance of the bloom period for infection and that the apparent effects on host susceptibility may therefore depend on the availability of specific FAs or combinations thereof.

Appressoria-Small but Incredibly Powerful Structures in Plant-Pathogen Interactions.

Plant-pathogenic fungi are responsible for many of the most severe crop diseases in the world and remain very challenging to control. Improving current protection strategies or designating new measures based on an overall understanding of molecular host-pathogen interaction mechanisms could be helpful for disease management. The attachment and penetration of the plant surface are the most important events among diverse plant-fungi interactions. Fungi evolved as small but incredibly powerful infection structure appressoria to facilitate attachment and penetration. Appressoria are indispensable for many diseases, such as rusts, powdery mildews, and blast diseases, as well as devastating oomycete diseases. Investigation into the formation of plant-pathogen appressoria contributes to improving the understanding of the molecular mechanisms of plant-pathogen interactions. Fungal host attachment is a vital step of fungal pathogenesis. Here, we review recent advances in the molecular mechanisms regulating the formation of appressoria. Additionally, some biocontrol agents were revealed to act on appressorium. The regulation of fungal adhesion during the infective process by acting on appressoria formation is expected to prevent the occurrence of crop disease caused by some pathogenic fungi.

SsCak1 Regulates Growth and Pathogenicity in Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is a devastating fungal pathogen that causes severe crop losses worldwide. It is of vital importance to understand its pathogenic mechanism for disease control. Through a forward genetic screen combined with next-generation sequencing, a putative protein kinase, SsCak1, was found to be involved in the growth and pathogenicity of S. sclerotiorum. Knockout and complementation experiments confirmed that deletions in SsCak1 caused defects in mycelium and sclerotia development, as well as appressoria formation and host penetration, leading to complete loss of virulence. These findings suggest that SsCak1 is essential for the growth, development, and pathogenicity of S. sclerotiorum. Therefore, SsCak1 could serve as a potential target for the control of S. sclerotiorum infection through host-induced gene silencing (HIGS), which could increase crop resistance to the pathogen.

The C2H2 Transcription Factor SsZFH1 Regulates the Size, Number, and Development of Apothecia in Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is a notorious phytopathogenic Ascomycota fungus with a host range of >600 plant species worldwide. This homothallic Leotiomycetes species reproduces sexually through a multicellular apothecium that produces and releases ascospores. These ascospores serve as the primary inoculum source for disease initiation in the majority of S. sclerotiorum disease cycles. The regulation of apothecium development for this pathogen and other apothecium-producing fungi remains largely unknown. Here, we report that a C2H2 transcription factor, SsZFH1 (zinc finger homologous protein), is necessary for the proper development and maturation of sclerotia and apothecia in S. sclerotiorum and is required for the normal growth rate of hyphae. Furthermore, ΔSszfh1 strains exhibit decreased H2O2 accumulation in hyphae, increased melanin deposition, and enhanced tolerance to H2O2 in the process of vegetative growth and sclerotia formation. Infection assays on common bean leaves, with thin cuticles, and soybean and tomato leaves, with thick cuticles, suggest that the deletion of Sszfh1 slows the mycelial growth rate, which in turn affects the expansion of leaf lesions. Collectively, our results provide novel insights into a major fungal factor mediating maturation of apothecia with additional effects on hyphae and sclerotia development.

First Report of Powdery Mildew Caused by Podosphaera xanthii on Cuphea hyssopifolia (Lythraceae) in Mainland China.

Cuphea hyssopifolia (Mexican heather) is a popular evergreen perennial shrub used for ornamental and medicinal purposes. Due to its high ornamental value, it is often used as a ground cover in parks and gardens in China. During February and March 2019 & 2020, powdery mildew was observed on C. hyssopifolia in the districts of Minhou and Jinshan of Fuzhou, China. Disease incidence was 70% but of low severity with only a few older leaves showing yellowing and wilting. Sparse irregular patches of white superficial powdery mildew observed on both sides of mature and young leaves. The powdery mildew fungal appressoria that occurred on epigenous hyphae, were indistinct to nipple-shaped, hyaline, and smooth. Conidiophores were erect, smooth, 80 to 210 × 10 to 12 µm, and produced two to eight crenate-shaped conidia in chains. Foot-cells of conidiophores were straight, cylindric, and 30 to 65 × 10 to12 µm. Conidia were hyaline, smooth, ellipsoid-ovoid to barrel-shaped, 25 to 38 × 16 to 20 µm with distinct fibrosin bodies. Germ tubes were simple to forked and produced from the lateral position of the germinating conidia. No chasmothecia were observed on the surface of infected leaves. Based on the morphology of the imperfect state, the powdery mildew fungus was identified as Podosphaera xanthii (Castagne) U. Braun & N. Shishkoff (Braun and Cook 2012). To confirm fungal identification, total DNA was extracted (Mukhtar et al., 2018) directly from epiphytic mycelia on infected leaves collected from both districts. Internal transcribed spacer (ITS) regions and the partial large subunit (LSU) rDNA were amplified using primers ITS1/ITS4 and LSU1/LSU2 (Scholin et al. 1994, White et al. 1990), respectively. The sequences were deposited in GenBank (ITS: MW692364, MW692365; LSU: MW699924, MW699925). The ITS and LSU sequences were 99 to 100 % identical to those of P. xanthii in GenBank, (ITS: MT568609, MT472035, MT250855, and AB462800; LSU: AB936276, JX896687, AB936277, and AB936274). Koch's postulates were completed by gently pressing diseased leaves onto leaves of five healthy potted C. hyssopifolia plants that were held in a greenhouse at 24 to 30°C without humidity control. Five non-inoculated plants served as controls. Inoculated plants developed symptoms after 6 to 10 days, whereas the controls remained symptomless. The morphology of the fungus on the inoculated leaves was identical to that observed on the originally diseased leaves. Previously, Podosphaera sp. has been reported on C. rosea in the United Kingdom (Beales & Cook 2008) and P. xanthii on C. hyssopifolia in Taiwan (Yeh et al. 2021). To our knowledge, this is the first report of powdery mildew caused by P. xanthii on C. hyssopifolia in mainland China. Our field observations suggest that the P. xanthii infections would be a potential threat to the health of C. hyssopifolia in China. References: Beales, P. A., and Cook, R. T. A. 2008. Plant Pathol. 57:778. Braun, U., Cook, R. T. A. 2012. The Taxonomic Manual of the Erysiphales (Powdery Mildews). CBS Biodiversity Series 11: CBS. Utrecht, The Netherlands. Mukhtar, I., et al. 2018. Sydowia.70:155. Scholin, C. A., et al. 1994. J. Phycol. 30:999. White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA. Yeh, Y. W., et al. 2021. Trop. Plant Pathol. 46:44.

Dynamic assembly of a higher-order septin structure during appressorium morphogenesis by the rice blast fungus.

The rice blast fungus Magnaporthe oryzae differentiates a specialized infection structure called an appressorium, which is used to break into plant cells by directed application of enormous turgor force. Appressorium-mediated plant infection requires timely assembly of a higher-order septin ring structure at the base of the appressorium, which is needed to spatially orchestrate appressorium repolarization. Here we use quantitative 4D widefield fluorescence imaging to gain new insight into the spatiotemporal dynamics of septin ring formation, and septin-mediated actin re-organization, during appressorium morphogenesis by M. oryzae. We anticipate that the new knowledge will provide a quantitative framework for dissecting the molecular mechanisms of higher-order septin ring assembly in this devastating plant pathogenic fungus.

Appressoria and phosphorus fluxes in mycorrhizal plants: connections between soil- and plant-based hyphae.

Arbuscular mycorrhizal fungi (AMF) live in symbiosis with plant roots, facilitating mineral nutrient transfer from soil to hosts through large networks of extraradical hyphae. Limited data are available on the fungal structures (appressoria) connecting soil- to root-based mycelium, in relation to plant nutrition. Two in vivo systems were set up using three AMF, Funneliformis mosseae, Funneliformis coronatus and Rhizoglomus irregulare, grown in symbiosis with Cichorium intybus. The assessment of plant P content, number of appressoria, diameter of their subtending hyphae and length of colonized roots allowed calculation of the total cross-section area of appressorium-subtending hyphae, which differed among the three AMF and was correlated with plant P contents and with extraradical mycelium density. A conservative evaluation of P fluxes from soil- to plant-based hyphae occurring through appressoria gave values ranging from 1.7 to 4.2 × 10-8 mol cm-2 s-1 (moles per total cross-section area of the appressorium subtending hyphae per time elapsed), depending on AMF identity. This work suggests that, beyond intraradical colonization and extraradical mycelium extent, connections between extraradical and intraradical fungal mycelium through appressoria are important for mycorrhizal plant nutrition, as appressorium structural traits and density can be related to P transfer mediated by AMF.

A Feed-Forward Subnetwork Emerging from Integrated TOR- and cAMP/PKA-Signaling Architecture Reinforces Magnaporthe oryzae Appressorium Morphogenesis.

Appressoria are important mediators of plant-microbe interactions. In the devastating rice blast pathogen Magnaporthe oryzae, appressorial morphogenesis from germ tube tips requires activated cAMP/PKA signaling and inactivated TOR signaling (TORoff). TORoff temporarily arrests G2 at a metabolic checkpoint during the single round of mitosis that occurs following germination. G2 arrest induces autophagy and appressorium formation concomitantly, allowing reprogression of the cell cycle to G1/G0 quiescence and a single appressorial nucleus. Inappropriate TOR activation abrogates G2 arrest and inhibits cAMP/PKA signaling downstream of cPKA. This results in multiple rounds of germ tube mitosis and the loss of autophagy and appressoria formation. How cAMP/PKA signaling connects to cell cycle progression and autophagy is not known. To address this, we interrogated TOR and cAMP/PKA pathways using signaling mutants, different surface properties, and specific cell cycle inhibitors and discovered a feed-forward subnetwork arising from TOR- and cAMP/PKA-signaling integration. This adenylate cyclase-cAMP-TOR-adenylate cyclase subnetwork reinforces cAMP/PKA-dependent appressorium formation under favorable environmental conditions. Under unfavorable conditions, the subnetwork collapses, resulting in reversible cell cycle-mediated germ tube growth regardless of external nutrient status. Collectively, this work provides new molecular insights on germ tube morphogenetic decision-making in response to static and dynamic environmental conditions.

Specialized infection strategies of falcate and oval conidia of Colletotrichum graminicola.

For many filamentous fungi with pathogenic lifestyles, the presence of distinct asexual conidia has been described. However, the role of these spore types remains mostly obscure. Colletotrichum graminicola is a hemibiotrophic filamentous fungus, causing anthracnose on maize plants with a high potential of epidemic disease spreading. C. graminicola generates two types of conidia. Falcate shaped conidia formed in necrotic lesions on maize tissues are able to generate appressoria with high efficiency and are considered key disease spreading propagules. The second conidia type, the smaller oval conidia, is formed in the vascular system of the infected plant, probably causing the distribution of the disease in planta. Barely any knowledge exists about how these conidia are able to exhibit their specific functions in the life cycle and pathogenicity of C. graminicola. Here, we show that germlings derived from both falcate and oval conidia differ in the secretion of a germination inhibitor and signals for germling fusion. Germination experiments combined with HPLC and mass spectrometry analyses revealed that germination of falcate conidia is regulated by the self-inhibitor mycosporine-glutamine, whereas this compound is absent from oval conidia cultures. Additionally, germlings derived from oval conidia undergo germling fusions at high frequencies and are able to induce such a fusion when co-incubated with falcate conidia. Falcate conidia germlings alone, however, were never observed to fuse. Plant infection experiments showed a positive correlation between germling fusions and efficient leaf infection by oval conidia. However, this correlation was not observed for infection by falcate conidia. Together, our findings reveal significant differences of two types of conidia derived from the same pathogenic fungus with distinct roles in pathogenesis.

Role of membrane compartment occupied by Can1 (MCC) and eisosome subdomains in plant pathogenicity of the necrotrophic fungus Alternaria brassicicola.

BACKGROUND: MCC/eisosomes are membrane microdomains that have been proposed to participate in the plasma membrane function in particular by regulating the homeostasis of lipids, promoting the recruitment of specific proteins and acting as provider of membrane reservoirs. RESULTS: Here we showed that several potential MCC/eisosomal protein encoding genes in the necrotrophic fungus A. brassicicola were overexpressed when germinated spores were exposed to antimicrobial defence compounds, osmotic and hydric stresses, which are major constraints encountered by the fungus during the plant colonization process. Mutants deficient for key MCC/eisosome components did not exhibit any enhanced susceptibility to phytoalexins and to applied stress conditions compared to the reference strain, except for a slight hypersensitivity of the ∆∆abpil1a-abpil1b strain to 2 M sorbitol. Depending on the considered mutants, we showed that the leaf and silique colonization processes were impaired by comparison to the wild-type, and assumed that these defects in aggressiveness were probably caused by a reduced appressorium formation rate. CONCLUSIONS: This is the first study on the role of MCC/eisosomes in the pathogenic process of a plant pathogenic fungus. A link between these membrane domains and the fungus ability to form functional penetration structures was shown, providing new potential directions for plant disease control strategies.

Surface sensing and signaling networks in plant pathogenic fungi.

Pathogenic fungi have evolved highly varied and remarkable strategies to invade and infect their plant hosts. Typically, such fungal pathogens utilize highly specialized infection structures, morphologies or cell types produced from conidia or ascospores on the cognate host surfaces to gain entry therein. Such diverse infection strategies require intricate coordination in cell signaling and differentiation in phytopathogenic fungi. Here, we present an overview of our current understanding of cell signaling and infection-associated development that primes host penetration in the top ten plant pathogenic fungi, which utilize specific receptors to sense and respond to different surface cues, such as topographic features, hydrophobicity, hardness, plant lipids, phytohormones, and/or secreted enzymes. Subsequently, diverse signaling components such as G proteins, cyclic AMP/Protein Kinase A and MAP kinases are activated to enable the differentiation of infection structures. Recent studies have also provided fascinating insights into the spatio-temporal dynamics and specialized sequestration and trafficking of signaling moieties required for proper development of infection structures in phytopathogenic fungi. Molecular insight in such infection-related morphogenesis and cell signaling holds promise for identifying novel strategies for intervention of fungal diseases in plants.

The appressorium of the rice blast fungus Magnaporthe oryzae remains mitotically active during post-penetration hyphal growth.

To investigate the mitotic dynamics of an appressorium, we used live-cell confocal imaging of a fluorescence-based mitotic reporter strain of Magnaporthe oryzae. We present evidence that the M. oryzae appressorium remains viable and mitotically active well after host penetration. These results suggest the potential roles of the appressorium during post-penetration proliferation of invasive hyphae. Our studies also revealed that a mitotic appressorial nucleus undergoes extreme constriction and elongation as it migrates through the penetration peg in a manner analogous to mitosis during cell-to-cell movement of invasive hyphae. Understanding the mechanisms underlying these pathogen-specific nuclear dynamics may provide new targets for disease control.

Colletotrichum higginsianum extracellular LysM proteins play dual roles in appressorial function and suppression of chitin-triggered plant immunity.

The genome of the hemibiotrophic anthracnose fungus, Colletotrichum higginsianum, encodes a large repertoire of candidate-secreted effectors containing LysM domains, but the role of such proteins in the pathogenicity of any Colletotrichum species is unknown. Here, we characterized the function of two effectors, ChELP1 and ChELP2, which are transcriptionally activated during the initial intracellular biotrophic phase of infection. Using immunocytochemistry, we found that ChELP2 is concentrated on the surface of bulbous biotrophic hyphae at the interface with living host cells but is absent from filamentous necrotrophic hyphae. We show that recombinant ChELP1 and ChELP2 bind chitin and chitin oligomers in vitro with high affinity and specificity and that both proteins suppress the chitin-triggered activation of two immune-related plant mitogen-activated protein kinases in the host Arabidopsis. Using RNAi-mediated gene silencing, we found that ChELP1 and ChELP2 are essential for fungal virulence and appressorium-mediated penetration of both Arabidopsis epidermal cells and cellophane membranes in vitro. The findings suggest a dual role for these LysM proteins as effectors for suppressing chitin-triggered immunity and as proteins required for appressorium function.

Simultaneous transcriptome analysis of Colletotrichum gloeosporioides and tomato fruit pathosystem reveals novel fungal pathogenicity and fruit defense strategies.

The fungus Colletotrichum gloeosporioides breaches the fruit cuticle but remains quiescent until fruit ripening signals a switch to necrotrophy, culminating in devastating anthracnose disease. There is a need to understand the distinct fungal arms strategy and the simultaneous fruit response. Transcriptome analysis of fungal-fruit interactions was carried out concurrently in the appressoria, quiescent and necrotrophic stages. Conidia germinating on unripe fruit cuticle showed stage-specific transcription that was accompanied by massive fruit defense responses. The subsequent quiescent stage showed the development of dendritic-like structures and swollen hyphae within the fruit epidermis. The quiescent fungal transcriptome was characterized by activation of chromatin remodeling genes and unsuspected environmental alkalization. Fruit response was portrayed by continued highly integrated massive up-regulation of defense genes. During cuticle infection of green or ripe fruit, fungi recapitulate the same developmental stages but with differing quiescent time spans. The necrotrophic stage showed a dramatic shift in fungal metabolism and up-regulation of pathogenicity factors. Fruit response to necrotrophy showed activation of the salicylic acid pathway, climaxing in cell death. Transcriptome analysis of C. gloeosporioides infection of fruit reveals its distinct stage-specific lifestyle and the concurrent changing fruit response, deepening our perception of the unfolding fungal-fruit arms and defenses race.

Screening of a synthetic peptide combinatorial library to identify inhibitors of the appressorium formation in Magnaporthe oryzae.

The rice blast disease caused by Magnaporthe oryzae is one of the most devastating diseases of cultivated rice. One of the most important stages in the infective cycle of M. oryzae is the formation of the dome-shaped structure called appressorium. The purpose of the present study was to identify novel peptides to control the rice blast disease by blocking the appressorium formation through screening of a synthetic peptide combinatorial library. As result of the screening, a set of 29 putative bioactive peptides were identified, synthesized and assayed in comparison with the previously identified peptide PAF104. The peptides MgAPI24, MgAPI40 and MgAPI47 showed improved inhibitory activity on the M. oryzae appressorium formation. Our data show that these peptides have a differential effect on two developmental structures: appressoria and appressorium-like structures. Antimicrobial assays against M. oryzae and other non-target microorganisms showed a weak or no toxicity of these peptides, demonstrating their specific activity blocking the appressorium formation. Therefore, the outcome of this research would be useful in the development of novel target-oriented peptides to use in plant protection.