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Citrus is one of the most important fruit crops in Mediterranean countries such as Spain, which is one of the main citrus-producing countries worldwide. Soil-borne pathogens, such as Rosellinia necatrix, are relevant limiting biotic factors in fruit trees, due to their tricky management. This fungus is a polyphagous plant pathogen with worldwide distribution, causing white root rot in woody crops, including citrus trees in Spain. The objective of this study was to evaluate the tolerance of new plant material against R. necatrix infection. Therefore, plants of 12 different citrus rootstocks were inoculated with one R. necatrix isolate. During the assay, and periodically, above-ground symptoms and chlorophyll content were evaluated. At the end of the experiment, leaf area and plant biomass measures were obtained. Rootstocks B11R5T64 and B11R5T60 achieved the lowest disease incidence of symptoms and reduction of biomass, and were similar to their respective controls in chlorophyll content and leaf area. Carrizo citrange, CL-5146 and UFR-5 were the most affected rootstocks in symptoms and biomass reduction. This work provides information about R. necatrix-tolerant citrus rootstocks, which can constitute a new integrated, sustainable and effective long-term strategy to avoid white root rot., This research was funded by the research contracts of Agromillora Catalana.

Rosellinia necatrix is the causal agent of white root rot (WRR), a fatal disease affecting many woody plants, including avocado (Persea americana). As with other root diseases, an integrated approach is required to control WRR. No fully effective control methods are available, and no chemical or biological agents against R. necatrix have been registered for use on avocado in South Africa. Fluazinam has shown promising results in the greenhouse and field in other countries, including Spain. The current study aimed to investigate the potential of a fumigant, chloropicrin, and biological control agents (B-Rus, Beta-Bak, Mity-Gro, and Trichoderma) against R. necatrix both in vitro and in vivo as compared with fluazinam. In a greenhouse trial, results showed that Trichoderma and B-Rus were as effective as fluazinam at inhibiting R. necatrix in vitro and suppressed WRR symptoms when applied before inoculation with R. necatrix. In contrast, Mity-Gro and Beta-Bak failed to inhibit the pathogen in vitro and in the greenhouse trial, despite application of the products to plants before R. necatrix infection. Fluazinam suppressed WRR symptoms in plants when applied at the early stages of infection, whereas chloropicrin rendered the pathogen nonviable when used as a preplant treatment. Plants treated with Trichoderma, B-Rus, and fluazinam sustained dry mass production and net CO2 assimilation by maintaining the green leaf tissues despite being infected with the pathogen. This study has important implications for the integrated management of WRR.

The current social demand for organic, sustainable, and eco-friendly approaches for farming, while ensuring the health and productivity of crops is increasing rapidly. Biocontrol agents are applied to crops to ensure biological control of plant pathogens. Research on the biological control of white root rot disease caused by a soil-borne pathogen, Rosellinia necatrix, is limited in pears compared to that in apple and avocado. This pathogenic fungus has an extensive host range, and symptoms of this disease include rotting of roots, yellowing and falling of leaves, wilting, and finally tree death. The severity of the disease caused by R. necatrix , makes it the most harmful fungal pathogen infecting the economical fruit tree species, such as pears, and is one of the main limiting factors in pear farming, with devastating effects on plant health and yield. In addition to agronomic and cultural practices, growers use chemical treatments to control the disease. However, rising public concern about environmental pollution and harmful effects of chemicals in humans and animals has facilitated the search for novel and environmentally friendly disease control methods. This review will briefly summarize the current status of biocontrol agents, ecofriendly methods, and possible approaches to control disease in pear orchards.

Esta enfermedad, provocada por el hongo Rosellinia necatrix Prill. (fa. Dematophora necatrix Hartig.), afecta a cerca de 170 especies vegetales. Si bien la enfermedad está mencionada en todo el mundo, aparentemente es más dañina en aquellas zonas con clima templado cálido y subtropical. El patógeno se caracteriza por ser un habitante del suelo. EEA La Consulta Fil: Pizzuolo, Pablo. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias; Argentina Fil: Lucero, Gabriela S. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias; Argentina

Avocado embryogenic cultures were selected for resistance to the culture filtrate (CF) of Rosellinia necatrix, the causal agent of White Root Rot disease. A resistant callus line was obtained through recurrent selections in progressively increasing concentrations of fungal CF (from 60% to 80%). RNA sequencing (RNA-Seq) technology was used to compare the transcriptomic profiles of the avocado embryogenic-callus-resistant line L3 (capable to survive in the presence of 80% CF) and control line AN-9 (not exposed to CF), after 24 h of growth in a medium containing 40% CF. A total of 25,211 transcripts were obtained, of which 4,918 and 5,716 were differentially expressed in the resistant and control line, respectively. Interestingly, exposure of embryogenic callus lines to 40% of R. necatrix exudates induced genes previously reported to be related to avocado defense against fungal diseases (lignin biosynthesis, Pathogenesis Related (PR) proteins, WRKY (WRKYGQK) Transcription Factor (TF), NAC (NAM, ATAF1/2, and CUC2) TF, proteinase inhibitors and Ethylene Response Transcription Factor (ERF), among others), which were accumulated in greater amounts in the resistant line in comparison to the susceptible one. This research will contribute to the understanding of avocado defense against this pathogen, thereby aiding in the selection of resistant avocado rootstocks.

Virus populations of uncultivated fungi remain scarcely studied. In the present study, we characterized a new partitivirus isolated from the false morel mushroom Gyromitra esculenta, named "Gyromitra esculenta partitivirus 1" (GePV1). The complete genome of GePV1, whose sequence was determined by combining high-throughput sequencing and RLM-RACE approaches, comprises two dsRNA segments of 1971 bp and 1799 bp, respectively. Each dsRNA genome segment contains a single open reading frame (ORF), encoding a putative RNA-dependent RNA polymerase (RdRp) and a capsid protein (CP), respectively. The sequences of the RdRp and CP exhibited the highest similarity (69.77% and 47.00% identity, respectively) to those of Rosellinia necatrix partitivirus 2 (RnPV2). Phylogenetic analysis based on the CP and RdRp sequences demonstrated that GePV1 clusters within a clade that includes members of the genus Alphapartitivirus, family Partitiviridae. We propose that GePV1 is a new member of the genus Alphapartitivirus. This is the first study reporting on a new partitivirus identified in the false morel mushroom Gyromitra esculenta.

Mycoviruses widely exist in diverse lineages of fungi, yet there are only a few studies on mycovirus infection in uncultivated fungi. We here report the presence of a dsRNA mycovirus in saprotrophic spring orange peel fungus Caloscypha fulgens. A novel dsRNA virus, named "Caloscypha fulgens fusagravirus 1" (CfFV1), was isolated from a single ascocarp of C. fulgens, and its molecular features were revealed. The full-length cDNA of CfFV1 comprises 9,548 nucleotides with a calculated GC content of 47.9% and two discontinuous open reading frames (ORF 1 and 2). A-1 ribosomal frameshift region with two distinctive elements, including a canonical slippery heptanucleotide (AAAAAAC) and a pseudoknot structure, predicted as a Recoding Stimulatory Element, was detected in the junction region of ORF1 and ORF2. The deduced amino acid sequence of ORF1 and ORF2 showed the highest similarity to the putative structural protein and RNA-dependent RNA polymerase (RdRp) of Rosellinia necatrix fusagravirus 4 (RnFGV4). Genome organization, sequence similarity, and phylogenetic analysis indicate that this virus belongs to a new member of the proposed family Fusagraviridae. This is the first report of the presence of a mycovirus in the spring orange peel fungus C. fulgens. Keywords: mycovirus; dsRNA; proposed Fusagraviridae; uncultivated fungi; Caloscypha fulgens.

We report here the presence of dsRNA mycoviruses in a Korean isolate of Rosellinia necatrix. A multiple band pattern of double-stranded RNA (dsRNA) from R. necatrix suggested mixed mycovirus infection. Next-generation sequencing analysis of purified dsRNAs indicated the presence of two dsRNA mycoviruses related to the members of families "Fusagraviridae" (proposed) and Partitiviridae. The first dsRNA virus revealed that the complete genome sequence was 8868 bp in size and contained two large open reading frames (ORFs 1 and 2), overlapped by 22 bp containing a canonical (- 1) slippery heptanucelotide sequence of UUUAAAC. The deduced amino acid sequence of ORF1 and ORF2 showed highest similarity to the hypothetical protein and RNA-dependent RNA polymerase (RdRp) of Rosellinia necatrix fusagravirus 3 (RnFGV3). Phylogenetic analysis showed that this dsRNA virus clustered with RnFGV3 and other fusagraviruses. Gene organization, sequence similarity, and phylogenetic analysis indicate that this virus seems to belong to a novel species of "Fusagraviridae", which we have named Rosellinia necatrix fusagravirus 4. The second virus has two dsRNA segments with sizes of 1907 bp and 1918 bp, each of which encoded a single ORF showing highest similarity to the RdRp and capsid protein of known members of Partitiviridae. Evaluation of genome structure, sequence similarity, and phylogeny indicate this to be a new member of the genus Alphapartitivirus in the family Partitiviridae, hereafter designated as Rosellinia necatrix partitivirus 26. This is the first report of the presence of a fusagravirus in an Asian R. necatrix isolate and of its mixed infection with a partitivirus.

Many Mediteranean isolates of Rosellinia necatrix, causing root rot in avocado, have previously been screened for viruses. Among them is isolate Rn459 that has been shown to be infected by at least three viruses such as Rosellinia necatrix partitivirus 10 (RnPV10), Rosellinia necatrix hypovirus 2 (RnHV2), Rosellinia necatrix fusagravirus 1 (RnFGV1), and Rosellinia necatrix virga-like virus (RnVLV). Here, we attempted to eliminate the viruses by hyphal tip cultures to examine their effect on colony growth in vitro and virulence on avocado plants. The obtained fungal strain termed, Rn459_PV10F/VLVF, which was confirmed to be cured of RnPV10 and RnVLV, but still retaining RnHV2, manifested a phenotype different from the original Rn459. Colony growth comparison showed that Rn459_PV10F/VLVF grew faster than the original Rn459 isolate and the virulence on avocado plants of this Rn459_PV10F/VLVF strain was higher than the original Rn459 strain. These combined results suggest that RnPV10 and RnVLV, alone or together, contribute to confer hypovirulence on the R. necatrix isolates., This study was supported in part by the Plan Nacional I + D + I Ministerio de Economía y Competitividad (AGL 2014–52,518-C2–2-R) Spain. In addition, this research was co-financed by FEDER funds (EU) and by Yomogi Inc. and Grantsin-Aid for Scientific Research on Innovative Areas from the Japanese Ministry of Education, Culture, Sports, Science and Technology (KAKENHI 25252011 and 16H06436, 16H06429 and 16 K21723 to N.S. and H. K.).

White root rot (WRR) disease caused by Rosellinia necatrix, a fungal pathogen, results in severe damage to various fruit trees, decreasing their marketability. Regular monitoring is a major process because the pathogen can remain in the soil around the host for a long time. Loop-mediated isothermal amplification (LAMP) is a highly sensitive and efficient amplification technology of nucleic acids (DNA or RNA) that can be performed at constant temperatures. Thus, it has been spotlighted as a useful tool for detecting several infectious agents. In the present study, LAMP-based Turn-on Fluorescent Paper (ToFP) devices were designed and applied to detect R. necatrix. LAMP conditions were optimized and found to be optimal at a reaction temperature (62 °C) and a reaction time (30 minutes). These reaction conditions were confirmed by applying them to infectious soil samples collected from the field. The limitation of detection was identified as 10 fg of genomic DNA under optimized LAMP conditions. These LAMP-based ToFP devices were generated with easily available stationery materials and the utility of these devices to analyze the LAMP results were confirmed through several experiments on a total of 14 field samples. The results showed that the developed LAMP-based detection system was very sensitive and had the advantages of rapid detection and high availability in the field.

La respuesta observada a nivel molecular en las plantas de aguacate mostró que durante el estrés hídrico se indujeron genes relacionados con otros tipos de estrés, tanto abiótico como biótico, destacando entre ellos genes que se expresan en genotipos de aguacate tolerantes a Rosellinia necatrix, el hongo patógeno causante de la Podredumbre Blanca Radicular (PBR) y que representa otro de los grandes problemas del cultivo de aguacate en climas mediterráneos. Asimismo, tras sufrir un estrés hídrico moderado se observó la permanencia de ciertos mecanismos de protección del aparato fotosintético y la sobreexpresión de determinados genes de defensa como el factor de transcripción NAC, endoquitinasas, PR-4, PR-5 o NPR1, induciendo en las plantas un estado de prealerta (“priming”). En comparación con plantas no estresadas o sometidas a estrés hídrico severo, las plantas que habían sufrido un estrés hídrico moderado mostraron un retaso en la progresión de la enfermedad al ser inoculadas con R. necatrix, sugiriendo que el estrés hídrico indujo una mayor tolerancia frente al ataque posterior de éste patógeno. Estos resultados, además de las implicaciones prácticas que puedan tener para el establecimiento de umbrales de humedad del suelo para el manejo del riego, abren la puerta a la utilización del estrés hídrico moderado como estrategia de manejo integrado frente a la PBR, cuya erradicación, una vez aparece en una plantación, es muy complicada. España es el primer productor de aguacate en Europa, con más de 15000 hectáreas plantadas en 2020 y una producción de 99125 toneladas. Más del 80% de esta producción se concentra en la denominada Costa Subtropical Andaluza, una zona que abarca parte de las provincias de Granada y Málaga y que presenta un clima de temperaturas suaves que, además de aguacate, permite el cultivo de otros frutales como el mango o chirimoyo. El aguacate es una fruta saludable, rica en vitaminas, minerales y grasas cuyo consumo se ha disparado a nivel mundial en la última década, por lo que su cultivo no deja de crecer en España. Sin embargo, las escasas precipitaciones del sur peninsular unidas al cambio climático que prevé episodios de sequía cada vez más extremos, ponen en duda el futuro de este frutal en la región. La presente tesis doctoral aborda tres grandes cuestiones fundamentales para la viabilidad y sostenibilidad del cultivo de aguacate en el sur peninsular; como son la determinación de las necesidades hídricas reales; la tolerancia y respuesta a diferentes estrategias de riego deficitario y, por último, la ventaja que conlleva la aplicación de determinadas estrategias de riego deficitario en el control de la respuesta del cultivo al patógeno Rosellinia necatrix. Con el fin de determinar la capacidad de las plantas de aguacate para hacer frente a la sequía, se ha llevado a cabo la caracterización de la respuesta fisiológica, mediante mediciones como el intercambio gaseoso y la fluorescencia de la hoja, y el análisis de la expresión génica, mediante la técnica de microarray y RT-qPCR de genes seleccionados, del portainjerto de aguacate ‘Dusa’, el más usado a nivel mundial, frente a distintos niveles de disponibilidad de agua. Este estudio ha aportado nuevos conocimientos sobre los mecanismos que se desencadenan en respuesta al estrés hídrico, así como sobre la capacidad de recuperación de ‘Dusa’ tras el mismo.

[Background] White root rot disease caused by Rosellinia necatrix is one of the most important threats affecting avocado productivity in tropical and subtropical climates. Control of this disease is complex and nowadays, lies in the use of physical and chemical methods, although none have proven to be fully effective. Detailed understanding of the molecular mechanisms underlying white root rot disease has the potential of aiding future developments in disease resistance and management. In this regard, this study used RNA-Seq technology to compare the transcriptomic profiles of R. necatrix during infection of susceptible avocado ‘Dusa’ roots with that obtained from the fungus cultured in rich medium., [Results] The transcriptomes from three biological replicates of R. necatrix colonizing avocado roots (RGA) and R. necatrix growing on potato dextrose agar media (RGPDA) were analyzed using Illumina sequencing. A total of 12,104 transcripts were obtained, among which 1937 were differentially expressed genes (DEG), 137 exclusively expressed in RGA and 160 in RGPDA. During the root infection process, genes involved in the production of fungal toxins, detoxification and transport of toxic compounds, hormone biosynthesis, gene silencing and plant cell wall degradation were overexpressed. Interestingly, 24 out of the 137 contigs expressed only during R. necatrix growth on avocado roots, were predicted as candidate effector proteins (CEP) with a probability above 60%. The PHI (Pathogen Host Interaction) database revealed that three of the R. necatrix CEP showed homology with previously annotated effectors, already proven experimentally via pathogen-host interaction., [Conclusions] The analysis of the full-length transcriptome of R. necatrix during the infection process is suggesting that the success of this fungus to infect roots of diverse crops might be attributed to the production of different compounds which, singly or in combination, interfere with defense or signaling mechanisms shared among distinct plant families. The transcriptome analysis of R. necatrix during the infection process provides useful information and facilitates further research to a more in -depth understanding of the biology and virulence of this emergent pathogen. In turn, this will make possible to evolve novel strategies for white root rot management in avocado., This research was supported by RTA2017–00040–00-00 (INIA-AEI), AVA201601.14 and AVA2019.008 (20% Junta de Andalucía, 80% FEDER) as well as AGL2017–83368-C2–1-R (Ministerio de Ciencia e Innovación) grants. C Pliego is currently supported by an INIA-CCAA contract, co-financed by INIA (20%) and FEDER (80%).

Rosellinia necatrix Prill. the causal agent of white root rot diseases causes major losses in avocado crops in Spain. To study the biocontrol of this disease, 31 antagonistic isolates were collected from the avocado rhizosphere and identified as Entoleuca sp. Studies were carried out in mycovirus detection, in vitro optimal growth temperature, mycelial compatibility groups of these isolates, and their inhibition of R. necatrix growth. The pathogenicity of these antagonistic isolates and their biocontrol of white root rot on avocado plants were also studied. The presence of mycoviruses was detected in all 31 Entoleuca sp. isolates studied and the optimal growth temperature for almost all the isolates (> 80%) was 25 °C. Dual culture showed different responses, with some isolates compatible and others incompatible with each other, showing inhibition halos and black lines. Twenty-five of the 31 isolates showed significant growth inhibition to a virulent R. necatrix isolate in in vitro conditions. In addition, none of the Entoleuca sp. isolates produced any symptoms on inoculated avocado plants and in 24 of them, symptoms of avocado white root rot were reduced, with significant differences among the Entoleuca sp. isolates when they were used as biocontrol agents. New virus-carrying Entoleuca sp. isolates were obtained, which are capable of controlling avocado white root rot., This study was partly supported by the Plan Nacional I+D+I Ministerio de Economía y Competitividad (AGL 2014-52518-C2-2-R) Spain. In addition, this research was co-financed by FEDER funds (EU).

White root rot disease, caused by Rosellinia necatrix, poses a threat to several tree crops; hence, effective and sustainable strategies to control this disease remain warranted. This study identified an effective R. necatrix biocontrol agent by isolating 32 strains from soil samples collected from white root rot-infested organic pear orchards, among which RDA1 exhibited the most potent growth-inhibitory effects. Microbiological and 16S rRNA gene sequencing analyses revealed that the bacterial isolate belonged to the Bacillus genus and exhibited 100% nucleotide sequence similarity with Bacillus velezensis species in the GenBank. This strain showed strong antifungal activity against four Rosellinia necatrix strains and harbored genes essential for lipopeptide, polyketide, and tripeptide bacilysin biosynthesis. RDA1 produced volatile compounds that suppressed the development of phytopathogens and possessed plant growth-promoting traits, such as phosphate solubilization, and indole-3-acetic acid and siderophore production. B. velezensis RDA1 has a significant potential application in sustainable agriculture and can be used to suppress white root rot disease infections and to improve plant growth.

The rhizobacterium Pseudomonas alcaligenes AVO110 exhibits antagonism toward the phytopathogenic fungus Rosellinia necatrix. This strain efficiently colonizes R. necatrix hyphae and is able to feed on their exudates. Here, we report the complete genome sequence of P. alcaligenes AVO110. The phylogeny of all available P. alcaligenes genomes separates environmental isolates, including AVO110, from those obtained from infected human blood and oyster tissues, which cluster together with Pseudomonas otitidis. Core and pan-genome analyses showed that P. alcaligenes strains encode highly heterogenic gene pools, with the AVO110 genome encoding the largest and most exclusive variable region (~1.6 Mb, 1795 genes). The AVO110 singletons include a wide repertoire of genes related to biofilm formation, several of which are transcriptionally modulated by R. necatrix exudates. One of these genes (cmpA) encodes a GGDEF/EAL domain protein specific to Pseudomonas spp. strains isolated primarily from the rhizosphere of diverse plants, but also from soil and water samples. We also show that CmpA has a role in biofilm formation and that the integrity of its EAL domain is involved in this function. This study contributes to a better understanding of the niche-specific adaptations and lifestyles of P. alcaligenes, including the mycophagous behavior of strain AVO110.

A method was developed to evaluate soil antagonism against the white root rot fungus, Rosellinia necatrix. Toothpicks colonized by the fungus were immersed into hot water to produce an extinction zone (EZ) of the fungus and then inserted into soil from different orchards. The EZ differed to various extents in unsterile soils; however, when soil was sterilized, the EZ disappeared. Using this method, we investigated the mechanism of the hot water drip irrigation method devised to control white root rot. The results showed the involvement of synergism between hot water and soil antagonists in the control measure against R. necatrix.

White root rot, caused by the soilborne fungus Rosellinia necatrix, is an important constraint to production for a wide range of woody crop plants such as avocado trees. The current methods of detection of white root rot are based on microbial and molecular techniques, and their application at orchard scale is limited. In this study, physiological parameters provided by imaging techniques were analyzed by machine learning methods. Normalized difference vegetation index (NDVI) and normalized canopy temperature (canopy temperature - air temperature) were tested as predictors of disease by several algorithms. Among them, logistic regression analysis (LRA) trained on NDVI data showed the highest sensitivity and lowest rate of false negatives. This algorithm based on NDVI could be a quick and feasible method to detect trees potentially affected by white root rot in avocado orchards., This work was supported by a grant from Consejería de Economía, Innovación, Ciencia y Empleo, Junta de Andalucía (P12-AGR-0370) to M. Barón, Ministerio de Economía, Industria y Competitividad Consejo Superior de Investigaciones Científicas and European Regional Development Fund (RECUPERA 2020/20134R060) to M. Barón, and Agencia de Innovación y Desarrollo de Andalucía (AGL14-52518-C2-1-R). C. Vida was recipient of a Ph.D. fellowship from the FPI program (MINECO).

Four isolates of Entoleuca sp., family Xylariaceae, Ascomycota, recovered from avocado rhizosphere in Spain were analyzed for mycoviruses presence. For that, the dsRNAs from the mycelia were extracted and subjected to metagenomics analysis that revealed the presence of eleven viruses putatively belonging to families Partitiviridae, Hypoviridae, Megabirnaviridae, and orders Tymovirales and Bunyavirales, in addition to one ourmia-like virus plus other two unclassified virus species. Moreover, a sequence with 98% nucleotide identity to plant endornavirus Phaseolus vulgaris alphaendornavirus 1 has been identified in the Entoleuca sp. isolates. Concerning the virome composition, the four isolates only differed in the presence of the bunyavirus and the ourmia-like virus, while all other viruses showed common patterns. Specific primers allowed the detection by RT-PCR of these viruses in a collection of Entoleuca sp. and Rosellinia necatrix isolates obtained from roots of avocado trees. Results indicate that intra- and interspecies horizontal virus transmission occur frequently in this pathosystem., This research was funded by the Spanish Ministry of Science and Innovation project AGL2014-52518-C2-2-R and the IFAPA projects AVA201301.13 and AVA201601.14, co-financed by FEDER.

Our study investigated the available chlorine content, contact time and difference among strains of each pathogen for sodium hypochlorite (NaOCl) to control chemically against soil-borne fungal pathogens, such as Phytophthora rot by Phytophthora cactorum, violet root rot by Helicobasidium mompa, and white root rot by Rosellinia necatrix, causing die-back symptom on apple trees. As a result, the colony growth of Phytophthora cactorum was inhibited completely by soaking over 5 s in 31.25 ml/l available chlorine content of NaOCl. Those of H. mompa and R. necatrix were inhibited entirely by soaking over 160 s in 62.5 and 125 ml/l available chlorine content in NaOCl, respectively. Also, inhibition effect on available chlorine in NaOCl among strains of each soil-borne pathogen showed no significant difference and was similar to or better than that of fungicides.

Rosellinia necatrix and Armillaria mellea are becoming serious problems in several Italian sweet cherry growing areas. In February 2005, a selection study was initiated at CREA-Centro di Ricerca per la Frutticoltura, based on 550 one-year-old seedlings of commercial myrobalan rootstocks (Prunus cerasifera L.), in order to identify promising myrobalan genotypes, in terms of agronomical, phenological, and productive characteristics. After micropropagation of the 42 best-performing seedlings, 16 were grafted with commercial cultivars, 'Burlat', 'Ferrovia', 'Giorgia', and 'Lapins'. In 2010, these were planted in a field trial at the field station in Rome, with MaxMa 60 (Prunus mahaleb × Prunus avium) serving as a reference rootstock. Here, the results obtained after 7 years of observations in the field are presented. A wide range of genetic variability was identified, in terms of flowering and ripening time, growth vigour and habit, suckering activity, productivity, and qualitative aspects of the fruits. Seven selections were identified as particularly interesting; these are being further evaluated for their resistance to the above-mentioned root rot diseases.

Rosellinia necatrix causes white root rot in various plants, including the Japanese pear. PCR assays using specific primers for R. necatrix detected the fungus on the roots of nine weed species from infested pear orchards. The soil inoculation experiment revealed that the spread of R. necatrix was similar between weed-mowed and non-weed-mowed treatments under field conditions. The spread of R. necatrix was also observed when rescue grass (Bromus catharticus) was grown in planter boxes under greenhouse conditions, but was limited without the grass, suggesting that some weeds facilitate the spread of R. necatrix in soil.

Industrial hemp (Cannabis sativa L.) was cultivated in Italy until the end of the Second World War. Since then, it has been abandoned and substituted with other crops mainly due to legal restrictions and public concerns. Public legislation passed in 2016, has allowed for the production of hemp seeds, flowers and fibers (law n. 242/2016). During a 2019 survey on hemp sanitary status in the province of Naples (40°57'6"12 N, 14°22'37"56 E), hemp 'Kompolty' with symptoms of root rot were observed at a private farm and collected for further analysis at the phytosanitary laboratory of CREA in Caserta. Death generally occurred within 2-3 weeks after the appearance of the first symptoms, occurring on ca. 10% of plants, consisting of yellowing, canopy wilt and signs of roots covered with white mycelium and fan-like mycelium under the bark. The causal agent, was isolated from small root segments, excised from symptomatic plants, the surface was disinfected with 2% sodium hypochlorite, placed on potato dextrose agar (PDA) amended with streptomycin sulphate (100mg/L) and incubated in the dark at 25°C for 5 days. Small pieces (2-3 mm) at the edge of the resulting colonies were sub-cultured onto PDA and incubated at 25°C in the dark for one week. The mycelia from 15 isolates showed pear-shaped swellings adjacent to the septa. The conidia were aseptate, hyaline, ellipsoid to ovoid, and 3-5 × 2.5-3 µm (n=50). Based on the morphological characteristics, the fungus was identified as Rosellinia necatrix Berl. ex Prill. (Singleton et al., 1992) a fungus taxonomically revised to Dematophora necatrix R. Hartig (Wittstein et al., 2020). To confirm the identification, total DNA was extracted from five isolates using a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and the ITS spacer was PCR-amplified with primers ITS1-ITS4 (White et al., 1990). The size-expected amplicons of 536 bp were purified and sequenced, the resulting sequence was trimmed and deposited in GenBank under the accession number MK937913. BLAST-n analysis revealed 98.83% nucleotide identity with some representative isolates of D. necatrix (MK888684.1; KT343972.1). To fulfill Koch's postulates, the pathogenicity tests were carried out on fifteen 4-weeks-old potted hemp plants 'Kompolty'. The inoculation was performed by adding 3 g of millet seeds inoculated with ten mycelial plugs, taken from the margins of a D. necatrix actively growing colony, per liter of sterile peat and perlite substrate in single pots. Moreover, ten hemp plants were inoculated with sterilized millet seed and served as negative controls. All plants were incubated at 25°C. After three weeks, inoculated plants exhibited foliar chlorosis, apical wilting, and death in two weeks, similar to what was observed in the field. Control plants did not show any symptoms. The fungus was isolated from the roots in all fifteen inoculated plants and confirmed to be D. necatrix based on morphological and molecular analysis, carried out with a second primer pair EF1-983F/ EF1-2218R targeting the transcription elongation factor 1- (Rehner and Buckley., 2005) (MW541068) that showed 99.67% nt in BLAST-n analysis. To our knowledge, this is the first report of D. necatrix infecting hemp in Europe. The farm where the problem arose has a history of cultivation for the production of apples for over 30 years. Therefore, an adaptation of D. necatrix to the new host is hypothesized. An in-depth knowledge on the diseases of hemp will be needed to relaunch hemp cultivation in this area.

Avocado (Persea americana) is an economically important fruit crop world-wide, the production of which is challenged by notable root pathogens such as Phytophthora cinnamomi and Rosellinia necatrix. Arguably the most prevalent, P. cinnamomi, is a hemibiotrophic oomycete which causes Phytophthora root rot, leading to reduced yields and eventual tree death. Despite its’ importance, the development of molecular tools and resources have been historically limited, prohibiting significant progress toward understanding this important host-pathogen interaction. The development of a nested qPCR assay capable of quantifying P. cinnamomi during avocado infection has enabled us to distinguish avocado rootstocks as either resistant or tolerant - an important distinction when unraveling the defense response. This review will provide an overview of our current knowledge on the molecular defense pathways utilized in resistant avocado rootstock against P. cinnamomi. Notably, avocado demonstrates a biphasic phytohormone profile in response to P. cinnamomi infection which allows for the timely expression of pathogenesis-related genes via the NPR1 defense response pathway. Cell wall modification via callose deposition and lignification have also been implicated in the resistant response. Recent advances such as composite plant transformation, single nucleotide polymorphism (SNP) analyses as well as genomics and transcriptomics will complement existing molecular, histological, and biochemical assay studies and further elucidate avocado defense mechanisms.

La rizobacteria Pseudomonas alcaligenes AVO110 muestra antagonismo frente al hongo fitopatógeno Rosellinia necatrix. Esta cepa coloniza eficazmente las hifas de R. necatrix y es capaz de alimentarse de sus exudados. Recientemente, hemos publicado la secuencia completa del genoma de P. alcaligenes AVO110. La filogenia de todos los genomas de P. alcaligenes disponibles separa los aislados ambientales, procedentes de agua, junto a la cepa AVO110, de los obtenidos de infecciones de sangre humana y de tejidos de ostras, que agrupan con Pseudomonas otitidis. Análisis del core y del pangenoma de P. alcaligenes han revelado que las cepas de esta especie codifican conjuntos de genes altamente heterogéneos, y que el genoma de AVO110 codifica la región variable más grande y exclusiva de todos ellos (aproximadamente 1,6 Mb y 1795 genes). Los genes exclusivos de AVO110 incluyen un amplio repertorio de genes relacionados con la formación de biopelículas (biofilms), de entre los cuales destacan aquellos modulados transcripcionalmente por exudados de R. necatrix. Uno de estos genes (cmpA) codifica una proteína con dominios GGDEF/EAL específica de cepas de Pseudomonas spp. aisladas de la rizosfera de diversas plantas, de suelo y de agua. También hemos demostrado que CmpA tiene un papel en la formación de biopelículas y que la integridad de su dominio EAL está involucrada en esta función. Este trabajo contribuye a una mejor comprensión del estilo de vida y de la adaptación a nichos específicos de P. alcaligenes, incluido el comportamiento micofágico de la cepa AVO110. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.

Pseudomonas chlororaphis PCL1606 (PcPCL1606) displays plant-colonizing features and exhibits antagonistic traits against soil-borne phytopathogenic fungi. Biofilm formation could be relevant for the PcPCL1606 lifestyle, and in this study the role of some putative extracellular matrix components (EMC; Fap-like fibre, alginate and Psl-like polysaccharides) in the biofilm architecture and biocontrol activity of this bacterium were determined. EMC such as the Fap-like fibre and alginate polysaccharide play secondary roles in biofilm formation in PcPCL1606, because they are not fundamental to its biofilm architecture in flow cell chamber, but synergistically they have shown to favour bacterial competition during biofilm formation. Conversely, studies on Psl-like polysaccharide have revealed that it may contain mannose, and that it is strongly involved in the PcPCL1606 biofilm architecture and niche competition. Furthermore, the Fap-like fibre and Psl-like exopolysaccharide play roles in early surface attachment and contribute to biocontrol activity against the white root rot disease caused by Rosellinia necatrix in avocado plants. These results constitute the first report regarding the study of the extracellular matrix of the PcPCL1606 strain and highlight the importance of a putative Fap-like fibre and Psl-like exopolysaccharide produced by PcPCL1606 in the biofilm formation process and interactions with the host plant root.

Biocontrol bacteria can be used for plant protection against some plant diseases. Pseudomonas chlororaphis PCL1606 (PcPCL1606) is a model bacterium isolated from the avocado rhizosphere with strong antifungal antagonism mediated by the production of the compound 2-hexyl, 5-propil resorcinol (HPR). Additionally, PcPCL1606 has biological control against different soil-borne fungal pathogens, including Rosellinia necatrix, which causes the emerging white root rot disease of many woody crops, including avocado in the Mediterranean area. The objective of this study was to assess whether the semicommercial application of PcPCL1606 to soil can potentially affect avocado soil and rhizosphere microbial communities and their multiple functions in natural conditions and under R. necatrix infection. To test the potential effects of PcPCL1606 on soil eukaryotic and prokaryotic communities, a formulated PcPCL1606 was prepared and applied to the soil of avocado plants growing in mesocosm experiments, and the communities were analysed by using 16S/ITS metagenomics. PcPCL1606 survived until the end of the experiments. The effect of PcPCL1606 application on prokaryotic communities in soil and rhizosphere samples from natural soil was not detectable, and very minor changes were observed in eukaryotic communities. In the infested soils, the presence of R. necatrix strongly impacted the soil and rhizosphere microbial communities. However, after PcPCL1606 was applied to soil infested with R. necatrix, the prokaryotic community reacted by increasing the relative abundance of few families with protective features against fungal soilborne pathogens and organic matter decomposition (Chitinophagaceae, Cytophagaceae), but no new prokaryotic families were detected. The treatment of PcPCL1606 impacted the fungal profile, which strongly reduced the presence of R. necatrix in avocado soil and rhizosphere, minimizing its effect on the rest of the microbial communities. The bacterial treatment of formulated PcPCL1606 on avocado soils infested with R. necatrix resulted in biological control of the pathogen. This suppressiveness phenotype was analysed, and PcPCL1606 has a key role in suppressiveness induction; in addition, this phenotype was strongly dependent on the production of HPR.

Partitiviruses (dsRNA viruses, family Partitiviridae) are ubiquitously detected in plants and fungi. Although previous surveys suggested their omnipresence in the white root rot fungus, Rosellinia necatrix, only a few of them have been molecularly and biologically characterized thus far. We report the characterization of a total of 20 partitiviruses from 16 R. necatrix strains belonging to 15 new species, for which “Rosellinia necatrix partitivirus 11–Rosellinia necatrix partitivirus 25” were proposed, and 5 previously reported species. The newly identified partitiviruses have been taxonomically placed in two genera, Alphapartitivirus, and Betapartitivirus. Some partitiviruses were transfected into reference strains of the natural host, R. necatrix, and an experimental host, Cryphonectria parasitica, using purified virions. A comparative analysis of resultant transfectants revealed interesting differences and similarities between the RNA accumulation and symptom induction patterns of R. necatrix and C. parasitica. Other interesting findings include the identification of a probable reassortment event and a quintuple partitivirus infection of a single fungal strain. These combined results provide a foundation for further studies aimed at elucidating mechanisms that underly the differences observed., This work was supported in part by Yomogi Inc. (to NS), the Program for the Promotion of Basic and Applied Research for Innovation in Bio-Oriented Industries (to SK and NS), and Grants-in-Aid for Scientific Research (A) and on Innovative Areas from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (MEXT) (17H01463 and 16H06436, 16H06429, and 16K21723 to NS).

A facile green route has been employed for the synthesis of ZnO and Ag-doped ZnO using Cannabis sativa as a reducing and stabilizing agent. The as-synthesized nanoparticles were characterized and tested for photocatalytic dye degradation and antimicrobial activity. The results suggested that nanoparticles have shown antimicrobial activity against different human pathogenic bacteria (Escherichia coli, Klebsiella pneumonia, MRSA, Pseudomonas aeruginosa, Salmonella typhi, Staphylococcus aureus) and fungal strains (Fusarium spp. and Rosellinia necatrix). Ag-doped nanoparticles comparatively have shown better removal Congo red and methyl orange under visible light. Therefore, green synthesized nanoparticles could have beneficial applications in environmental science and biological field.

Different studies to control the Rosellinia necatrix growth in vitro and avocado white root rot in vivo were performed, using two non-pathogenic R. necatrix isolates (Rn12 and Rn29) combined with fluazinam. The application of non-pathogenic isolates alone showed significant growth reduction as compared to R. necatrix pathogenic isolates in a PDA growth medium. The combination of both non-pathogenic isolates plus fluazinam at 0.01 mg l−1 significantly increased the reduction of the pathogen growth in vitro. Additionally, the combination of the lowest fluazinam concentration (0.001%) plus Rn12 improved control of the disease as compared when either the same fungicide rate was used alone or this isolate was applied alone on plants, showing a similar response to the use of the highest concentration of fluazinam applied alone (0.01%). In this work, we have obtained a new, integrated biological control method against this avocado disease using non-pathogenic R. necatrix isolates combined with low doses of fluazinam., This study was supported partly by the Plan Nacional I + D + I Ministerio de Economía y Competitividad (AGL 2014–52518-C2-2-R), Spain. In addition, the research was co-financed by FEDER funds (EU).

Rosellinia necatrix is responsible for the white rot root disease of avocado in Southern Spain. Entoleuca sp. is a fungus isolated from roots of these same trees, but it is not pathogenic in avocado. Here, we describe two new species of partitiviruses detected in isolates of the avocado sympatric fungi Entoleuca sp. and R. necatrix, termed Entoleuca partitivirus 1 (EnPV1), genus Alphapartitivirus, and Entoleuca partitivirus 2 (EnPV2), genus Betapartitivirus. For both R. necatrix and Entoleuca sp., the dsRNA of the RdRp genomic segment of EnPV1 accumulates at a higher rate than the CP dsRNA, except for a set of Entoleuca sp. isolates where titers of the CP dsRNA are 35–50 times higher than those of the RdRp dsRNA and between 250–380 times higher than the CP dsRNA titers found in the rest of Entoleuca sp. and R. necatrix isolates. For EnPV2, the accumulation rates of the RdRp dsRNA in Entoleuca sp., is in most of the cases, higher than the CP dsRNA. In contrast, in R. necatrix isolates, EnPV2 dsRNA2 generally accumulates at a higher rate. Genetic analysis of the partitiviruses revealed that there is no apparent variation in the nucleotide sequences among the strains. RNA silencing of the partitiviruses appears to be limited in Entoleuca sp., as shown by small RNA sequencing. Finally, the investigation of the presence of these partitiviruses in a fungal collection revealed that they have no role in the pathogenicity of R. necatrix in avocado or in the avirulence of Entoleuca sp. in this host., This work was supported by IFAPA project AVA201601.14, co-financed by FEDER, and by the Spanish Ministry of Science and Innovation project AGL2014-52518-C2-2-R.M.J. Alcalá technical assistance is acknowledged.

Tesis Doctoral.-- Universidad de Córdoba. Programa de Doctorado: Ingeniería Agraria, Agroalimentaria, Forestal y de Desarrollo Rural Sostenible., [EN] Spain is the highest avocado producer country in the European Union. White root rot caused by the ascomycete Rosellinia necatrix is one of the most important diseases of this crop in the southern coastal area of Iberian Peninsula due to its difficult management. We studied different integrated control methods of this disease and its possible combinations. First, we detected and identified mycoviruses in R. necatrix isolates and their relation with the hypovirulence factor as a future virocontrol method. As well we obtained novel antagonistic fungi (Entoleuca sp. isolates) in which we analyzed mycoviruses presence, their growth under different temperatures, their vegetative compatibility groups and their biocontrol effect against the disease. We improved a qPCR technique for the detection and quantification of this pathogen from avocado orchards soil samples, thus we used this novel technique to assess the inoculum quantity of R. necatrix in the soil after the treatments of the fungicide fluazinam that we carried out in commercial orchards. Furthermore we performed an integrated control of this disease combining low concentrations of fluazinam with antagonist organisms such as rhizobacteria strains and/or non-pathogenic R. necatrix isolates. In this work we have studied all possible chemical and biological control methods against this disease nowadays, even some of them can be applied directly in field conditions., [ES] España es el mayor productor de aguacate en la Unión Europea. La podredumbre blanca radicular causada por el hongo ascomiceto Rosellinia necatrix Prill. es una de las enfermedades más importantes de este cultivo en la zona costera sur de la península Ibérica por su difícil manejo. Estudiamos diferentes métodos de control integrado de esta enfermedad y sus posibles combinaciones. Primero detectamos e identificamos micovirus infectando aislados de R. necatrix y su relación con el factor de hipovirulencia como un futuro método de virocontrol. También obtuvimos novedosos hongos antagonistas (aislados de Entoleuca sp.) en los que analizamos la presencia de micovirus, su crecimiento a diferentes temperaturas, sus grupos de compatibilidad vegetativa y su efecto de biocontrol contra la enfermedad. Mejoramos una técnica de qPCR para la detección y cuantificación de este patógeno en suelo a partir de muestras procedentes de fincas de aguacate, que fue utilizada como novedosa técnica para evaluar la cantidad de inóculo de R. necatrix presente en el suelo tras los tratamientos del fungicida fluazinam que realizamos en fincas comerciales para el control de la enfermedad. Además realizamos un control integrado de esta, combinando dosis bajas de fluazinam con organismos antagonistas como cepas de rizobacterias y/o aislados no patogénicos de R. necatrix. En este trabajo se han estudiado todos los posibles métodos de controles biológicos y químicos utilizables contra esta enfermedad hoy en día, pudiendo ser alguno de ellos de aplicación inmediata en campo.

The NPR1 gene encodes a key component of systemic acquired resistance (SAR) signaling mediated by salicylic acid (SA). Overexpression of NPR1 confers resistance to biotrophic and hemibiotrophic fungi in several plant species. The NPR1 gene has also been shown to be involved in the crosstalk between SAR signaling and the jasmonic acid-ethylene (JA/Et) pathway, which is involved in the response to necrotrophic fungi. The aim of this research was to generate transgenic olive plants expressing the NPR1 gene from Arabidopsis thaliana to evaluate their differential response to the hemibiotrophic fungus Verticillium dahliae and the necrotroph Rosellinia necatrix. Three transgenic lines expressing the AtNPR1 gene under the control of the constitutive promoter CaMV35S were obtained using an embryogenic line derived from a seed of cv. Picual. After maturation and germination of the transgenic somatic embryos, the plants were micropropagated and acclimated to ex vitro conditions. The level of AtNPR1 expression in the transgenic materials varied greatly among the different lines and was higher in the NPR1-780 line. The expression of AtNPR1 did not alter the growth of transgenic plants either in vitro or in the greenhouse. Different levels of transgene expression also did not affect basal endochitinase activity in the leaves, which was similar to that of control plants. Response to the hemibiotrophic pathogen V. dahliae varied with pathotype. All plants died by 50 days after inoculation with defoliating (D) pathotype V-138, but the response to non-defoliating (ND) strains differed by race: following inoculation with the V-1242 strain (ND, race 2), symptoms appeared after 44–55 days, with line NPR1-780 showing the lowest disease severity index. This line also showed good performance when inoculated with the V-1558 strain (ND, race 1), although the differences from the control were not statistically significant. In response to the necrotroph R. necatrix, all the transgenic lines showed a slight delay in disease development, with mean area under the disease progress curve (AUDPC) values 7–15% lower than that of the control., This investigation was funded by the Junta de Andalucía (Grant No. P11-AGR-7992) and by the Ministerio de Ciencia e Innovación of Spain and Feder European Union Funds (Grant No. AGL2017-83368-C2-1-R).

Two monoconidial isolates of Trichoderma atroviride Miller and one of Trichoderma virens Giddens and Foster were combined with the contact fungicide fluazinam to improve the in vitro inhibition of Rosellinia necatrix Prillieux and the control of avocado white root rot caused by this fungus. A fluazinam concentration of 0.01 mg L −1 was obtained as the most effective minimum concentration for in vitro combinations of antagonist-fungicide, significantly reducing (on average, by 41%) the growth of a representative collection of pathogen isolates. The combinations of one isolate of Trichoderma atroviride or virens , plus fluazinam at 0.01 mg L −1 showed a greater in vitro growth inhibition of R. necatrix (36 and 65%, respectively) than the use of fluazinam alone (23%). However, the inhibition of R. necatrix growth by the other T. atroviride plus fluazinam was less than 20% and not significantly different from the control plus fluazinam. The isolate of T. virens applied alone or with fluazinam at 0.01 mg L −1 induced growth inhibition (65%) of the pathogen similar to that induced by the control with fluazinam at a concentration of 0.05 mg L −1 . A fluazinam concentration of 0.01% has been confirmed as effective for controlling white root rot of avocado. Furthermore, the combination of fluazinam at 0.001% with Trichoderma improves significantly the effective control of avocado white root rot on infected plants in comparison with treatments with the fungicide or the antagonist alone. In this way, our results suggest that applications of these antagonist/fungicide combinations should be recommended in order to avoid the development of resistance to the fungicide by the pathogen and improve the control of avocado white root rot.

A Victorivirus was detected in isolate F16176 of the fungus Fusarium asiaticum, the causal agent of Fusarium head blight in China. The full genome sequence of the virus was sequenced and characterized. The complete cDNA sequence is 5,281 nucleotides long with 64.2% G + C content and contains two open reading frames (ORFs) that overlap at the pentanucleotide UAAUG. The two ORFs are predicted to encode coat protein (CP) and RNA-dependent RNA polymerase (RdRp), which are conserved among the dsRNA mycoviruses of the genus Victorivirus. Pairwise comparisons and phylogenetic analysis of the deduced amino acid sequences of RdRp indicated that this dsRNA mycovirus is a new virus belonging to the species Rosellinia necatrix victorivirus 1 in the family Totiviridae. This study is the first to report a full-length genomic sequence of a putative member of the genus Victorivirus in F. asiaticum.

Two isolates of Rosellinia necatrix (Rn118-8 and Rn480) have previously obtained from diseased avocado trees in commercial orchards of the coastal area in southern Spain. Rn118-8 and Rn480 have weak virulence on avocado plants, and are infected by R. necatrix hypovirus 2 (RnHV2). In this work, the possible biological effects of the hypovirus on R. necatrix were tested. First, RnHV2 was transmitted from each of Rn118-8 and Rn480 to a highly virulent, RnHV2-free isolate of R. necatrix (Rn400) through hyphal anastomosis, using zinc compounds which attenuate the mycelial incompatibility reactions and allow for horizontal virus transfer between vegetatively incompatible fungal strains. Next, we carried out an analysis of growth rate in vitro and a virulence test of these newly infected strains in avocado plants. We obtained five strains of Rn400 infected by RnHV2 after horizontal transmission, and showed some of them to have lower colony growth in vitro and lower virulence on avocado plants compared with virus-free Rn400. These results suggest that R. necatrix isolates infected by RnHV2 could be used as novel virocontrol agents to combat avocado white root rot., This study was supported in part by the Plan Nacional I + D + I Ministerio de Economía y Competitividad (AGL 2014-52518-C2-2-R) Spain and co-financed by FEDER funds (EU) and by Yomogi Inc. and Grants-in-Aid for Scientific Research (A) and Scientific Research on Innovative Areas from the Japanese Ministry of Education, Culture, Sports, Science and Technology (17H01463 and 16H06436, 16H06429 and 16K21723 to N.S.).

The soilborne pathogen Rosellinia necatrix causes white root rot, a serious disease of various trees, and is extremely difficult to control. In this study, using one-dimensional electrophoresis coupled with nanoliquid chromatography-electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI-Q-TOF-MS/MS), we identified 696 proteins from R. necatrix mycelium (KACC 40445) grown in liquid culture. In addition, 573 proteins were assigned to at least one gene ontology term including 26 functional groups. Most were related to catalytic activity in the molecular function category. This proteomic data set advances understanding of R. necatrix biology and will inform further investigations to manage white root rot using novel strategies.

To reveal mycovirus diversity, we conducted a search of as-yet-unexplored Mediterranean isolates of the phytopathogenic ascomycete Rosellinia necatrix for virus infections. Of seventy-nine, eleven fungal isolates tested RNA virus-positive, with many showing coinfections, indicating a virus incidence of 14%, which is slightly lower than that (approximately 20%) previously reported for extensive surveys of over 1000 Japanese R. necatrix isolates. All viral sequences were fully or partially characterized by Sanger and next-generation sequencing. These sequences appear to represent isolates of various new species spanning at least 6 established or previously proposed families such as Partiti-, Hypo-, Megabirna-, Yado-kari-, Fusagra- and Fusarividae, as well as a newly proposed family, Megatotiviridae. This observation greatly expands the diversity of R. necatrix viruses, because no hypo-, fusagra- or megatotiviruses were previously reported from R. necatrix. The sequence analyses showed a rare horizontal gene transfer event of the 2A-like protease domain between a dsRNA (phlegivirus) and a positive-sense, single-stranded RNA virus (hypovirus). Moreover, many of the newly detected viruses showed the closest relation to viruses reported from fungi other than R. necatrix, such as Fusarium spp., which are sympatric to R. necatrix. These combined results imply horizontal virus transfer between these soil-inhabitant fungi.

White root rot disease, caused by the pathogen Rosellinia necatrix, is one of the world's most devastating plant fungal diseases and affects several commercially important species of fruit trees and crops. Recent global outbreaks of R. necatrix and advances in molecular techniques have both increased interest in this pathogen. However, the lack of information regarding the genomic structure and transcriptome of R. necatrix has been a barrier to the progress of functional genomic research and the control of this harmful pathogen. Here, we identified 10,616 novel full-length transcripts from the filamentous hyphal tissue of R. necatrix (KACC 40445 strain) using PacBio single-molecule sequencing technology. After annotation of the unigene sets, we selected 14 cell cycle-related genes, which are likely either positively or negatively involved in hyphal growth by cell cycle control. The expression of the selected genes was further compared between two strains that displayed different growth rates on nutritional media. Furthermore, we predicted pathogen-related effector genes and cell wall-degrading enzymes from the annotated gene sets. These results provide the most comprehensive transcriptomal resources for R. necatrix, and could facilitate functional genomics and further analyses of this important phytopathogen.

The improvement in soil quality of avocado crops through organic amendments with composted almond shells has a positive effect on crop yield and plant health, and enhances soil suppressiveness against the phytopathogenic fungus Rosellinia necatrix. In previous studies, induced soil suppressiveness against this pathogen was related to stimulation of Gammaproteobacteria, especially some members of Pseudomonas spp. with biocontrol-related activities. In this work, we isolated bacteria from this suppressiveness-induced amended soil using a selective medium for Pseudomonas-like microorganisms. We characterized the obtained bacterial collection to aid in identification, including metabolic profiles, antagonistic responses, hybridization to biosynthetic genes of antifungal compounds, production of lytic exoenzymatic activities and plant growth-promotion-related traits, and sequenced and compared amplified 16S rDNA genes from representative bacteria. The final selection of representative strains mainly belonged to the genus Pseudomonas, but also included the genera Serratia and Stenotrophomonas. Their biocontrol-related activities were assayed using the experimental avocado model, and results showed that all selected strains protected the avocado roots against R. necatrix. This work confirmed the biocontrol activity of these Gammaproteobacteria-related members against R. necatrix following specific stimulation in a suppressiveness-induced soil after a composted almond shell application.

Most double-stranded RNA (dsRNA) viruses are transcribed and replicated in a specialized icosahedral capsid with a T=1 lattice consisting of 60 asymmetric capsid protein (CP) dimers. These capsids help to organize the viral genome and replicative complex(es). They also act as molecular sieves that isolate the virus genome from host defense mechanisms and allow the passage of nucleotides and viral transcripts. Rosellinia necatrix quadrivirus 1 (RnQV1), the type species of the family Quadriviridae , is a dsRNA fungal virus with a multipartite genome consisting of four monocistronic segments (segments 1 to 4). dsRNA-2 and dsRNA-4 encode two CPs (P2 and P4, respectively), which coassemble into ∼450-Å-diameter capsids. We used three-dimensional cryo-electron microscopy combined with complementary biophysical techniques to determine the structures of RnQV1 virion strains W1075 and W1118. RnQV1 has a quadripartite genome, and the capsid is based on a single-shelled T=1 lattice built of P2-P4 dimers. Whereas the RnQV1-W1118 capsid is built of full-length CP, P2 and P4 of RnQV1-W1075 are cleaved into several polypeptides, maintaining the capsid structural organization. RnQV1 heterodimers have a quaternary organization similar to that of homodimers of reoviruses and other dsRNA mycoviruses. The RnQV1 capsid is the first T=1 capsid with a heterodimer as an asymmetric unit reported to date and follows the architectural principle for dsRNA viruses that a 120-subunit capsid is a conserved assembly that supports dsRNA replication and organization. IMPORTANCE Given their importance to health, members of the family Reoviridae are the basis of most structural and functional studies and provide much of our knowledge of dsRNA viruses. Analysis of bacterial, protozoal, and fungal dsRNA viruses has improved our understanding of their structure, function, and evolution, as well. Here, we studied a dsRNA virus that infects the fungus Rosellinia necatrix , an ascomycete that is pathogenic to a wide range of plants. Using three-dimensional cryo-electron microscopy and analytical ultracentrifugation analysis, we determined the structure and stoichiometry of Rosellinia necatrix quadrivirus 1 (RnQV1). The RnQV1 capsid is a T=1 capsid with 60 heterodimers as the asymmetric units. The large amount of genetic information used by RnQV1 to construct a simple T=1 capsid is probably related to the numerous virus-host and virus-virus interactions that it must face in its life cycle, which lacks an extracellular phase.

[Aims] This study aims to develop and validate a new molecular method of detection and quantification of Rosellinia necatrix fungus in soil samples and compare it with conventional methods., [Methods] We collected 40 soil and root samples (one as negative control) from the soil around avocado trees. The root samples were checked for typical symptoms of R. necatrix and the pathogen was identified using the conventional method of plate culture. These results were then corroborated using a new molecular method of detection and quantification of R. necatrix in soil samples, and a duplex TaqMan qPCR protocol was designed that included an internal positive control to avoid the detection of false negatives., [Results] The molecular detection and quantification method was effective, sensitive and reliable for all 40 soil samples analysed, whereas, with traditional methods, the fungus was isolated in only 24 out of the 26 symptomatic roots from 40 avocado trees sampled. This improved methodology reduces the sample preparation time compared with previous studies, and provides a molecular tool for the reliable and accurate detection and quantification of R. necatrix in naturally infested avocado soils., [Conclusions] This technique could be applied for the rapid assessment of R. necatrix in soils at the pre-planting stage and evaluation of the efficacy of physical, chemical or biological control treatments., This study was partly supported by the Spanish Plan Nacional I + D + I Ministerio de Economía y Competitividad (AGL 2014-52518-C2-2-R). The research was also co-financed by FEDER funds (EU).

Background White root rot (WRR) disease caused by Rosellinia necatrix is one of the most important threats affecting avocado orchards in temperate regions. The eradication of WRR is a difficult task and environmentally friendly control methods are needed to lessen its impact. Priming plants with a stressor (biotic or abiotic) can be a strategy to enhance plant defense/tolerance against future stress episodes but, despite the known underlying common mechanisms, few studies use abiotic-priming for improving tolerance to forthcoming biotic-stress and vice versa (‘cross-factor priming’). To assess whether cross-factor priming can be a potential method for enhancing avocado tolerance to WRR disease, ‘Dusa’ avocado rootstocks, susceptible to R. necatrix, were subjected to two levels of water stress (mild-WS and severe-WS) and, after drought-recovery, inoculated with R. necatrix. Physiological response and expression of plant defense related genes after drought-priming as well as the disease progression were evaluated. Results Water-stressed avocado plants showed lower water potential and stomatal limitations of photosynthesis compared to control plants. In addition, NPQ and qN values increased, indicating the activation of energy dissipating mechanisms closely related to the relief of oxidative stress. This response was proportional to the severity of the water stress and was accompanied by the deregulation of pathogen defense-related genes in the roots. After re-watering, leaf photosynthesis and plant water status recovered rapidly in both treatments, but roots of mild-WS primed plants showed a higher number of overexpressed genes related with plant defense than severe-WS primed plants. Disease progression after inoculating primed plants with R. necatrix was significantly delayed in mild-WS primed plants. Conclusions These findings demonstrate that mild-WS can induce a primed state in the WRR susceptible avocado rootstock ‘Dusa’ and reveal that ‘cross-factor priming’ with water stress (abiotic stressor) is effective for increasing avocado tolerance against R. necatrix (biotic stressor), underpinning that plant responses against biotic and abiotic stress rely on common mechanisms. Potential applications of these results may involve an enhancement of WRR tolerance of current avocado groves and optimization of water use via low frequency deficit irrigation strategies.

Rosellinia necatrix causes white root rot in various plants, including the Japanese pear. PCR assays using specific primers for R. necatrix detected the fungus on the roots of nine weed species from infested pear orchards. The soil inoculation experiment revealed that the spread of R. necatrix was similar between weed-mowed and non-weed-mowed treatments under field conditions. The spread of R. necatrix was also observed when rescue grass (Bromus catharticus) was grown in planter boxes under greenhouse conditions, but was limited without the grass, suggesting that some weeds facilitate the spread of R. necatrix in soil.

Tiger nut (Cyperus esculentus L.) is cultivated in Spain for the production of tiger nut milk. Over the past 5 years, important economic losses resulting from a new tuber rot have been observed near Valencia in eastern Spain. Affected tubers were covered by a white mycelium that turned black as the disease advanced, leading to a general rotting of tubers. Aboveground plant parts showed some early decay and under high-moisture environmental conditions the white mycelium was present on the soil surface. This mycelium showed pyriform swellings characteristic of Rosellinia necatrix Prill., and coremia were occasionally observed on external surfaces of tubers after incubation in a moist chamber for 1 to 2 months. Coremia produced small ellipsoid or obovoid single-celled conidia 3.7 to 5.0 × 2.0 to 2.2 μm. The teleomorph has not been observed. Pathogenicity tests were conducted with inoculum produced on wheat (Triticum aestivum L.) seeds that were soaked for 12 h in flasks filled with distilled water. Each flask contained 300 ml of seeds that were subsequently autoclaved after excess water was drained. Two fungal disks of a 2-week-old culture of R. necatrix grown on potato dextrose agar were placed aseptically in each flask. The flasks were incubated at 25°C for 4 weeks, and shaken once a week to avoid clustering of inoculum. Two plastic pots (35 cm in diameter) per isolate were filled with a sterilized mixture of equal portions (vol/vol) of soil, sand, and peat moss, and inoculum was added at a concentration of 30 g of infected wheat seeds per 1,200 g of substrate (1). Healthy tubers were surface disinfested in 1.5% (vol/vol) sodium hypochlorite for 1 min, washed twice in sterile water, sown, and subsequently thinned to one per pot after emergence. Plants were grown under field conditions. Six months after inoculation, symptomatic tubers appeared similar to those originally observed in the field. The fungus was reisolated from affected tubers, confirming Koch's postulates. Pathogenicity tests were also conducted on avocado (cv. Reed) and almond (cv. Garriges) seedlings, and apple rootstock (MM-106) as very susceptible host plants for R. necatrix (1). Inoculated plants showed symptoms of wilting and death 4 weeks after inoculation. The fungus was reisolated from affected plants. This is the first report of C. esculentus as a host of R. necatrix. Reference: (1) A. Sztejnberg and Z. Madar. Plant Dis. 64: 662, 1980.

Rosellinia necatrix is the causal agent of avocado white root rot (WRR). Control of this soil-borne disease is difficult, and the use of tolerant rootstocks may present an effective method to lessen its impact. To date, no studies on the molecular mechanisms regulating the avocado plant response towards this pathogen have been undertaken. To shed light on the mechanisms underpinning disease susceptibility and tolerance, molecular analysis of the gene's response in two avocado rootstocks with a contrasting disease reaction was assessed. Gene expression profiles against R. necatrix were carried out in the susceptible 'Dusa' and the tolerant selection BG83 avocado genotypes by micro-array analysis. In 'Dusa', the early response was mainly related to redox processes and cell-wall degradation activities, all becoming enhanced after disease progression affected photosynthetic capacity, whereas tolerance to R. necatrix in BG83 relied on the induction of protease inhibitors and their negative regulators, as well as genes related to tolerance to salt and osmotic stress such as aspartic peptidase domain-containing proteins and gdsl esterase lipase proteins. In addition, three protease inhibitors were identified, glu protease, trypsin and endopeptidase inhibitors, which were highly overexpressed in the tolerant genotype when compared to susceptible 'Dusa', after infection with R. necatrix, reaching fold change values of 52, 19 and 38, respectively. The contrasting results between 'Dusa' and BG83 provide new insights into the different mechanisms involved in avocado tolerance to Phytophthora cinnamomi and R. necatrix, which are consistent with their biotrophic and necrotrophic lifestyles, respectively. The differential induction of genes involved in salt and osmotic stress in BG83 could indicate that R. necatrix penetration into the roots is associated with osmotic effects, suggesting that BG83's tolerance to R. necatrix is related to the ability to withstand osmotic imbalance. In addition, the high expression of protease inhibitors in tolerant BG83 compared to susceptible 'Dusa' after infection with the pathogen suggests the important role that these proteins may play in the defence of avocado rootstocks against R. necatrix.

The production of the compound 2-hexyl-5-propyl resorcinol (HPR) by the biocontrol rhizobacterium Pseudomonas chlororaphis PCL1606 (PcPCL1606) is crucial for fungal antagonism and biocontrol activity that protects plants against the phytopathogenic fungus Rosellinia necatrix. The production of HPR is also involved in avocado root colonization during the biocontrol process. This pleiotrophic response prompted us to study the potential role of HPR production in biofilm formation. The swimming motility of PcPLL1606 is enhanced by the disruption of HPR production. Mutants impaired in HPR production, revealed that adhesion, colony morphology, and typical air-liquid interphase pellicles were all dependent on HPR production. The role of HPR production in biofilm architecture was also analyzed in flow chamber experiments. These experiments revealed that the HPR mutant cells had less tight unions than those producing HPR, suggesting an involvement of HPR in the production of the biofilm matrix.

By transversely cutting infected avocado plant stems and using PCR techniques on avocado leaves, two experiments were carried out to determine whether Rosellinia necatrix can invade avocado vascular tissues. We were unable to detect the pathogen in either stems or leaves in either experiment, so we concluded that R. necatrix does not invade the vascular system of the plant. Additionally, the toxins produced by the pathogen were also studied to determine whether such toxins could contribute to the wilting and death of avocado plants infected by R. necatrix, having an effect on avocado leaves, where they can hinder the photosynthetic process. First, we isolated and identified the toxins cytochalasin E and rosnecatrone from filtrates of six R. necatrix isolates. Second, we tried to detect cytochalasin E in sap and leaves from infected avocado plants, and it was not detected at the minimum level of 50 μg/kg in leaves or 25 μg/kg on sap. Finally, we observed changes in fluorescence emitted by the avocado leaf surface (to detect photosynthetic efficiency) after inoculating avocado plants with this toxin. Fluorescence was higher in the leaves of plants immersed in toxin solution after 4 and 8 days, but not after longer periods of time. In this work, we demonstrated that although R. necatrix is not a fungus that invades the vascular system, its toxins are probably involved in the wilting and death of infected avocado plants, decreasing the efficiency of photosynthesis., This research was supported by the “Consejería de Innovación, Ciencia y Empresa” Junta de Andalucía grant (Grupo PAIDI AGR-235) and by the Plan Nacional I + D + I from Ministerio de Ciencia e Innovación (AGL 2011-030354-CO2-02). In addition, this research was co-financed by FEDER funds (EU).

Fungi are an important component of the soil ecosystem. Mycoviruses have numerous potential impacts on soil fungi, including phytopathogenic fungal species. However, the diversity and ecology of mycoviruses in soil fungi is largely unexplored. Our previous work has shown that the soil-borne phytopathogenic fungus Rosellinia necatrix was infected with several novel mycoviruses after growing for 2-3 years in an apple orchard. In this study, we investigated whether natural infection of R. necatrix with mycoviruses occurs under limited conditions. Virus-free R. necatrix isolates were grown in a small bucket containing soil samples for a short time (1.5-4.5 months) under greenhouse conditions. Screening of dsRNA mycoviruses among 365 retrieved isolates showed that four, including 6-31, 6-33, 6-35, and 7-11, harbored virus-like dsRNAs. Molecular characterization of the dsRNAs revealed that three retrieved isolates, 6-31, 6-33, and 6-35 were infected with a novel endornavirus and isolate 7-11 is infected with a novel partitivirus belonging to the genus Alphapartitivirus. These novel mycoviruses had no overt biological impact on R. necatrix. Overall, this study indicates that natural infections of R. necatrix with new mycoviruses can occur under experimental soil conditions.

The alternaria blackspot of rapeseed is one of the most prominent diseases of rapeseed. It is caused by three species of the genus Alternaria: Alternaria brassicicola, Alternaria brassicae, and Alternaria raphanin. Here we report a novel positive-sense RNA virus from an A. brassicicola strain 817-14. The virus has a 6639 nucleotide (nt) long genome, excluding a poly (A)-tail, and was predicted to contain three putative open reading frames (ORF1, ORF2, and ORF3). The large ORF1 encoded a 174-kDa polyprotein (composed of 1522 amino acid residues) containing a conserved RNA-dependent RNA polymerase (RdRp) domain and a helicase domain. The other two smaller ORFs encoded polypeptides with unknown function. Homology search and phylogenetic analysis, based on the RdRp and helicase domains, suggest that this virus is related to and grouped with Sclerotinia sclerotiorum fusarivirus 1 (SsFV1), Rosellinia necatrix fusarivirus 1 (RnFV1), Fusarium graminearum virus-DK21 (FgV1), and Penicillium roqueforti RNA mycovirus 1 (PrRV1), all of which belong to a newly proposed family Fusariviridae. For this study, we designed the virus as "Alternaria brassicicola fusarivirus 1" (AbFV1). Virus elimination revealed that AbFV1 has no conspicuous impact on the biological properties of its host.