Emerging roles of plant microRNAs during Colletotrichum spp. infection.

MAIN CONCLUSION: This review provides valuable insights into plant molecular regulatory mechanisms during fungus attacks, highlighting potential miRNA candidates for future disease management. Plant defense responses to biotic stress involve intricate regulatory mechanisms, including post-transcriptional regulation of genes mediated by microRNAs (miRNAs). These small RNAs play a vital role in the plant's innate immune system, defending against viral, bacterial, and fungal attacks. Among the plant pathogenic fungi, Colletotrichum spp. are notorious for causing anthracnose, a devastating disease affecting economically important crops worldwide. Understanding the molecular machinery underlying the plant immune response to Colletotrichum spp. is crucial for developing tools to reduce production losses. In this comprehensive review, we examine the current understanding of miRNAs associated with plant defense against Colletotrichum spp. We summarize the modulation patterns of miRNAs and their respective target genes. Depending on the function of their targets, miRNAs can either contribute to host resistance or susceptibility. We explore the multifaceted roles of miRNAs during Colletotrichum infection, including their involvement in R-gene-dependent immune system responses, hormone-dependent defense mechanisms, secondary metabolic pathways, methylation regulation, and biosynthesis of other classes of small RNAs. Furthermore, we employ an integrative approach to correlate the identified miRNAs with various strategies and distinct phases of fungal infection. This study provides valuable insights into the current understanding of plant miRNAs and their regulatory mechanisms during fungus attacks.

Quiescence as a strategic stage for the infective process of Colletotrichum species

Colletotrichum, a genus of Ascomycota fungi with a diversity of species grouped into several species complexes, or clades, is associated with diseases known as "Anthracnose". It affects significantly different tropical and subtropical fruit species. Infections occur in the field and postharvest; quiescent stages are mainly responsible for postharvest losses. An analysis of this pre-infective stage is made based on consulted papers. The infective process is modulated by the length of the quiescent period of the fungus during the vegetative or pre-productive stages of trees. Quiescent stage is determined by the host's biochemical responses and the pathogen's activity. Once the pathogen is activated, it develops an infectious necrotrophic process. Colletotrichum quiescence has been studied mainly in subtropical fruits and horticultural species, and the studies highlight the role of volatile compounds, metabolites and enzymes involved in the length and breaking of quiescence, as well as the differential responses according to the phenological stage and the genotype of the plant. Transcriptomic and proteomic analysis of the host-fungus interaction have revealed the role of genes in the occurrence and breaking of quiescence. Knowledge about the quiescence of Colletotrichum in tropical fruits is necessary to improve management efficiency. Detection and induction of quiescent infections has been studied and genomics has allowed to understand the occurrence of quiescence in the infective process; however, in crops in the tropics, such as mango, an important crop in Colombia, questions about associated species and biology of their quiescence are still unanswered.

Colletotrichum es un hongo ascomicete, con diversidad de especies agrupadas en complejos o clados y se asocia a enfermedades conocidas, como "Antracnosis" y afecta significativamente especies de frutas tropicales y subtropicales. Las infecciones ocurren en el campo y postcosecha; los estados quiescentes son los principales responsables de pérdidas en postcosecha. Se realizó un análisis de esta etapa pre-infectiva, a partir de publicaciones consultadas. La infección está modulada por la duración del período de quiescencia del hongo durante las etapas vegetativa o pre-productivas de los árboles y está determinado por las respuestas bioquímicas del hospedante y la actividad del patógeno. Una vez se activa el patógeno, se desarrolla un proceso infeccioso necrotrófico. La quiescencia de Colletotrichum se ha estudiado, principalmente, en frutas subtropicales y especies hortícolas y destacan el papel de compuestos volátiles, metabolitos y enzimas en la duración y en la pérdida de la quiescencia, así como las respuestas diferenciales, según la fenología y el genotipo. Análisis transcriptómicos y proteómicos de la interacción hospedante-hongo han revelado el papel de genes en la ocurrencia y pérdida de quiescencia. Conocer la quiescencia de Colletotrichum en frutas tropicales es necesario para hacer más eficiente el manejo de la enfermedad. Se ha estudiado la detección e inducción de infecciones quiescentes y estudios genómicos han permitido entender su ocurrencia durante la infección; sin embargo, en cultivos en el trópico, como el mango, un cultivo importante en Colombia, las preguntas sobre las especies asociadas y la biología de la quiescencia de estas, aún están sin respuesta.

PpCRN7 and PpCRN20 of Phythophthora parasitica regulate plant cell death leading to enhancement of host susceptibility.

BACKGROUND: Phytophthora species secrete cytoplasmic effectors from a family named Crinkler (CRN), which are characterised by the presence of conserved specific domains in the N- and C-terminal regions. P. parasitica causes disease in a wide range of host plants, however the role of CRN effectors in these interactions remains unclear. Here, we aimed to: (i) identify candidate CRN encoding genes in P. parasitica genomes; (ii) evaluate the transcriptional expression of PpCRN (Phytophthora parasitica Crinkler candidate) during the P. parasitica interaction with Citrus sunki (high susceptible) and Poncirus trifoliata (resistant); and (iii) functionally characterize two PpCRNs in the model plant Nicotiana benthamiana. RESULTS: Our in silico analyses identified 80 putative PpCRN effectors in the genome of P. parasitica isolate 'IAC 01/95.1'. Transcriptional analysis revealed differential gene expression of 20 PpCRN candidates during the interaction with the susceptible Citrus sunki and the resistant Poncirus trifoliata. We have also found that P. parasitica is able to recognize different citrus hosts and accordingly modulates PpCRNs expression. Additionally, two PpCRN effectors, namely PpCRN7 and PpCRN20, were further characterized via transient gene expression in N. benthamiana leaves. The elicitin INF-1-induced Hypersensitivity Response (HR) was increased by an additive effect driven by PpCRN7 expression, whereas PpCRN20 expression suppressed HR response in N. benthamiana leaves. Despite contrasting functions related to HR, both effectors increased the susceptibility of plants to P. parasitica. CONCLUSIONS: PpCRN7 and PpCRN20 have the ability to increase P. parasitica pathogenicity and may play important roles at different stages of infection. These PpCRN-associated mechanisms are now targets of biotechnological studies aiming to break pathogen's virulence and to promote plant resistance.

Microscopic analysis of colonization of Colletotrichum abscissum in citrus tissues.

Postbloom fruit drop (PFD), caused mainly by Colletotrichum abscissum, is one of the most severe citrus diseases and can causes up to 80% fruit loss in favorable climatic conditions. According to the literature, other Colletotrichum species colonize hosts using distinct strategies: intracellular hemibiotrophic or subcuticular intramural necrotrophic colonization. However, so far, for C. abscissum only the necrotrophic stage has been described and some aspects remain unclear in PFD disease cycle. To better understand the disease cycle, microscopy studies could be applied. However, even using eGFP strains (expressing green fluorescent protein), the results are unclear due to the autofluorescence of citrus leaves. To eliminate this problem and to study the interaction between C. abscissum-citrus we used a destaining and staining methodologies, and we observed that in leaves, even applying injury before inoculation, C. abscissum does not colonize adjacent tissues. Apparently, in the leaves the fungus only uses the nutrients exposed in the artificial lesions for growth, and then produces large amount of spores. However, in flowers, C. abscissum penetrated and colonized the tissues of the petals 12 h after inoculation. In the early stages of infection, we observed the development of primary biotrophic hyphae, suggesting this species as a hemibiotrophic fungus, with a short biotrophic phase during flower colonization followed by dominant necrotrophic colonization. In conclusion, the use of an eGFP strain of C. abscissum and a different methodology of destaining and staining allowed a better understanding of the morphology and mechanisms used by this citrus pathogen to colonize the host.

Water relation, leaf gas exchange and chlorophyll a fluorescence imaging of soybean leaves infected with Colletotrichum truncatum.

Considering the potential of anthracnose to decrease soybean yield and the need to gain more information regarding its effect on soybean physiology, the present study performed an in-depth analysis of the photosynthetic performance of soybean leaflets challenged with Colletotrichum truncatum by combining chlorophyll a fluorescence images with gas-exchange measurements and photosynthetic pigment pools. There were no significant differences between non-inoculated and inoculated plants in leaf water potential, apparent hydraulic conductance, net CO2 assimilation rate, stomatal conductance to water vapor and transpiration rate. For internal CO2 concentration, significant difference between non-inoculated and inoculated plants occurred only at 36 h after inoculation. Reductions in the values of the chlorophyll a fluorescence parameters [initial fluorescence (F0), maximal fluorescence (Fm), maximal photosystem II quantum yield (Fv/Fm), quantum yield of regulated energy dissipation (Y(NPQ))] and increases in effective PS II quantum yield (Y(II)), quantum yield of non-regulated energy dissipation Y(NO) and photochemical quenching coefficient (qP) were noticed on the necrotic vein tissue in contrast to the surrounding leaf tissue. It appears that the impact of the infection by C. truncatum on the photosynthetic performance of the leaflets was minimal considering the preference of the fungus to colonize the veins.

Caracterización morfológica y molecular de una colección de aislamientos de Phomopsis longicolla(teleomorfo desconocido: Diaporthales)de la región templada y subtropical de Argentina

Diaporthe(telomorfo) - Phomopsis(anamorfo) (DP) constituye un grupo fúngico de amplia diversidad genética con más de 900 especies distribuidas en un amplio rango de hospedantes que incluye especies cultivadas y no cultivadas, forestales, frutales y malezas. Los aislamientos de DP son hemi-biótrofos y disponen de diferentes fuentes de inóculo primario, como el rastrojo y las semillas, para reiniciar sus ciclos de parasitismo-saprofitismo. Ellos colonizan los tejidos del hospedante desde los estadios tempranos del desarrollo y establecen relaciones nutricionales de endofitia y necrotrofia fúngica. La plasticidad del género Phomopsisha favorecido su expansión a diferentes agro-ecosistemas y hospedantes constituyendo un importante riesgo epidemiológico. El objetivo fue validar la identidad y evaluar las relaciones biológicas de 12 aislamientos de P. longicollay D. phaseolorumvar. sojaeobtenidos en distintos agro-ambientes templados y subtropicales de Argentina, para analizar la variabilidad y estrategias de conservación de la bio-diversidad fúngica. Las cualidades macro-morfo-lógicas(textura y color de colonias, forma y distribución de estromas, desarrollo, forma y distribución de cuerpos fructíferos), y los caracteres micro-morfológicos(tamaño y forma de conidios, ascos y ascosporas) permitieron identificar a tres nuevos aislamientos de P. longicollaincluidos en el complejo D/P. El análisis molecular complementario corrigió las limitaciones derivadas de la caracterización basada sólo en marcadores morfológicos y logró reubicar al aislamiento AFP.8413 de identidad dudosa, en el nodo correspondiente a P. longicolla.De esta manera, la caracterización molecular definió la identidad de los aislamientos y los ubicó en los 4 taxones del complejo DP: diez aislamientos fueron incluidos en Plo(AFP.Gpo 4.4, AFP.Gpo 3.5, AFP.Gpo 4.3, AFP.Gpo 3.4, AFP.CaA, AFP. CaB, AFP.B5L16, AFP.B4L17, AFP.227B2, AFP.8413), un aislamiento incluido en Dps(AFP.Qcol7), un aislamiento en Dpc(AFP.Dpc16) y dos aislamientos incluidos en Dpm(AFP.Dpm109 y AFP.Dpm112). La adecuada identificación de P. longicollay el avance en el conocimiento de las relaciones biológicas (hibridaciones homo o heterotálicas) entre variedades de D. phaseolorum (P. phaseoli)y especies de Diaporthe- Phomopsispermiten comprender la plasticidad para colonizar un amplio rango de hospedantes, los mecanismos de variabilidad genética y la preservación de la diversidad fúngica.

Diaporthe(teleomorpho)-Phomopsis (anamorph) (DP) is a fungal group of great genetic diversity with over 900 species associated to a wide host range that includes cultivated and uncultivated species, forest, fruit trees and weeds. DP isolates are hemi-biotrophs and have different sources of primary inoculum as stubble and seeds to restart cycles of parasitism -saprophytism. They colonize host tissues from early plant stages and establish different nutritional relationships, acting as endophytic and necrotrophic fungi. The plasticity of the Phomopsisgenus has favored its expansion to different agro-ecosystems and various hosts constituting an epidemiological risk. The objective was to validate the identity and evaluate the biological relationships among 12 isolates of P. longicollaand D. phaseolorumvar. sojae(anamorph P. phaseolivar. sojae)obtained in different tempered and subtropical agro-environments of Argentina, in order to analyze the variability and strategies for preserving fungal biodiversity. Macro-morphological attributes (such as texture and color of colonies, stroma shape and distribution, pycnidia and perythecia shape and distribution) and micro-morphological characteristics (such as size and shape of conidia, asci and ascospores) allowed identifying three new isolates as P. longicolla.A complementary molecular analysis was also made to overcome the limitations derived from the morphological analysis, thus the AFP.8413 isolate was finally identified as P. longicolla.The molecular characterization was useful to identify the evaluated isolates and to group them in four taxa of the Diaporthe-Phomopsiscomplex: ten isolates were included in P. longicolla,one isolate was included in D. phaseolorumvar. sojae(anamorph P. phaseolivar. sojae),one isolate was identified as D. phaseolorumvar. caulivoraand two isolates were included in D. phaseolorumvar. meridi-onalis.The use of phenotipic and molecular tools have contributed to an accurate identification of P. longicolla,and comprehension about the biological relationships (homo or heterothallic hibridizations) among D. phaseolo-rumvarieties (P. phaseoli)and species of Diaporthe-Phomopsis.This allowed also a better understanding of the mechanisms of fungic plasticity, to colonize and expand their host range and genetic variability, promoting thus their biodiversity conservation.

Apoplastic and intracellular plant sugars regulate developmental transitions in witches' broom disease of cacao.

Witches' broom disease (WBD) of cacao differs from other typical hemibiotrophic plant diseases by its unusually long biotrophic phase. Plant carbon sources have been proposed to regulate WBD developmental transitions; however, nothing is known about their availability at the plant-fungus interface, the apoplastic fluid of cacao. Data are provided supporting a role for the dynamics of soluble carbon in the apoplastic fluid in prompting the end of the biotrophic phase of infection. Carbon depletion and the consequent fungal sensing of starvation were identified as key signalling factors at the apoplast. MpNEP2, a fungal effector of host necrosis, was found to be up-regulated in an autophagic-like response to carbon starvation in vitro. In addition, the in vivo artificial manipulation of carbon availability in the apoplastic fluid considerably modulated both its expression and plant necrosis rate. Strikingly, infected cacao tissues accumulated intracellular hexoses, and showed stunted photosynthesis and the up-regulation of senescence markers immediately prior to the transition to the necrotrophic phase. These opposite findings of carbon depletion and accumulation in different host cell compartments are discussed within the frame of WBD development. A model is suggested to explain phase transition as a synergic outcome of fungal-related factors released upon sensing of extracellular carbon starvation, and an early senescence of infected tissues probably triggered by intracellular sugar accumulation.