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A collection of Bacillus and Trichoderma isolates from Iran was investigated for the biological control of pear white root rot caused by Rosellinia necatrix. Among the isolates, B. amyloliquefaciens AP4, B. siamensis AP8 and T. harzianum T20a were selected based on their growth inhibition of R. necatrix in dual cultures, as well as by volatiles, diffusible extracts and culture filtrates. Four pear rootstocks (Pyrodwarf, OHF40, OHF60 and Williams) were assessed for white rot in the greenhouse following soil amendment with the three isolates. For OHF40 rootstock, none of the three isolates significantly reduced disease severity, whereas all three isolates significantly reduced disease severity for OHF69 rootstock. For Pyrodwarf rootstock, only T. harzianum T20a and B. amyloliquefaciens AP4 significantly reduced root rot, but all three isolates significantly reduced leaf fall. For Williams rootstock, all three isolates significantly reduced root rot, but only T. harzianum T20a significantly reduced leaf fall. Thus, the effectiveness of both the Bacillus and Trichoderma isolates was highly dependent upon the pear genotype. The lowest levels of white rot symptoms were observed in the Williams‐T. harzianum T20a interaction resulting in root rot severity of 46.6% and leaf fall of 4.7% at 70 dpi. While the effect of plant genotype on biocontrol activity has been reported frequently for Trichoderma, this is one of the first to report such an effect with a Bacillus biocontrol agent. Both Bacillus and Trichoderma can be effective biocontrol agents of pear white rot, but the plant genotype can have a major impact on their effectiveness. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Rosellinia necatrix (Prill.) is one of the most important soil-borne pathogens affecting fruit trees in Iran. A preliminary selection of open-pollinated seedlings of 13 Prunus spp. genotypes, including 'Early Golden', 'Obilnaja', 'Mirabolano', 'Blue Free', 'Tanasgol', 'Mandchurica', 'Saint Julien', 'Greengage', 'Cadaman', GF-677, K-R.T-02, K-R.T-01, and K-R.T-41, for resistance to white root rot disease was carried out through artificial inoculation in the greenhouse as part of plum and prune rootstocks breeding programme. In the first experiment, among 559 seedlings belonging to the Prunus population, 282 plants survived 120 days after inoculation. The survivors were exposed to twofold higher concentration of inoculum. The successful establishment of the pathogen on roots of 'Early Golden', 'Obilnaja', 'Greengage, K-R.T-41, 'Blue Free', and 'Tanasgol' led to the destruction of almost all plants. The resistance level of 'Mirabolano', 'Saint Julien', 'Blue Free', and K-R.T-02 did not change with the amount of inoculum, and they were defined as moderately resistant in both experiments. Among all studied Prunus spp. germplasm, the resistance level of the seedlings of K-R. T-01 was determined as resistant regardless of inoculum density. Although a large number of hybrid GF-677 seedlings died with exposure to a low level of inoculum, the remaining plants were able to survive even at high fungal density. Similarly, the plants of 'Mandchurica' and 'Cadaman' showed resistant responses with a high level of inoculum. It was a preliminary test for the screening of Prunus spp. population against R. necatrix. The selected seedlings that survived after two fungal inoculation trials are currently being evaluated for reproductive performance and will be screened for precise resistance to white root rot disease in the near future. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Rosellinia necatrix is an ascomycete that causes white root rot (WRR) of several plant host species resulting in economic losses to affected agricultural and forestry industries in various regions. This study aimed to identify and monitor the prevalence of R. necatrix in avocado orchards in South Africa. We used both morphological and molecular methods to isolate and identify R. necatrix from diseased plant material and soil. Results showed that R. necatrix was present on avocado in the Limpopo, Mpumalanga and KwaZulu-Natal provinces. Additionally, a semi-selective medium, containing Rose Bengal, nystatin, cycloheximide, chlorothalonil and 2-phenylphenol, was developed to improve isolation of R. necatrix. We also tested an already established R. necatrix-specific TaqMan qPCR protocol to determine if it can reliably detect the pathogen isolates in planta in the South African samples. Based on our results the technique had a detection rate of 91.3% in artificially infected roots and 100% in artificially inoculated soil. We tested natural infected plant and soil samples and detected R. necatrix in 86% of the plant samples and in 70% of the soil samples. Using a selective medium or an in planta molecular detection method streamlines isolation and detection of R. necatrix, which will help prevent further spread of the pathogen. Moreover, additional information on the prevalence of WRR will create awareness among growers and provide a basis for management of the disease. [ABSTRACT FROM AUTHOR]

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.

Three Trichoderma harzianum isolates, were evaluated for their antagonistic effect on Rosellinia necatrix causal agent of white root rot (WRT). According to in vitro evaluations, T. harzianum T20A isolate showed the most pathogen growth inhibition. The inoculum of T20A isolate was applied to control WRT disease on four commercial apple rootstocks: Malling (M7, M25) and Malling Merton (MM111, MM106) in greenhouse experiments. Root rot and leaf fall indices were measured 70 days after pathogen inoculation. The biocontrol agent had a significant effect (p < 0.01) on the reduction of pathogen indices but the effect of rootstocks was not significant. Root rot reduction on MM111, MM106, M25 and M7 rootstocks were 63.84%, 61.13%, 28.63% and 17.47%, respectively. The antagonist also caused reduction of leaf drop symptom on MM106, M7, MM111 and M25 infected apple rootstocks by 57.4%, 56.06%, 44.09% and 40.24%, respectively. Disease indices were also measured for fungicide treatment and the results were compared with disease indices in antagonist treatments. The most biological control was observed on MM111 (63.84%) and MM106 (57.4%) according to the reduction in root rot and leaf drop, respectively. The reactions of apple rootstocks to WRT were also evaluated. The results showed that all the rootstocks were susceptible to WRT in the greenhouse condition. The MM106 rootstock which showed 100% root rot and 78% leaf drop was the most susceptible and M7with 43.5% root rot and 84.56 leaf drop was the least susceptible in our experiment. This was the first study of reaction and biocontrol of white root rot disease on apple commercial rootstocks in Iran and the results suggest a better insight to disease management either by integrating resistance and biocontrol measures or replacing chemical control by antagonist application to soil.

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.

We previously proposed a new virus lifestyle or yadokari/yadonushi nature exhibited by a positive-sense single-stranded RNA (ssRNA) virus, yadokari virus 1 (YkV1), and an unrelated double-stranded RNA (dsRNA) virus, yadonushi virus 1 (YnV1) in a phytopathogenic ascomycete, Rosellinia necatrix. We have proposed that YkV1 diverts the YnV1 capsid to trans-encapsidate YkV1 RNA and RNA-dependent RNA polymerase (RdRp) and replicate in the heterocapsid. However, it remains uncertain whether YkV1 replicates using its own RdRp and whether YnV1 capsid copackages both YkV1 and YnV1 components. To address these questions, we first took advantage of the reverse genetics tools available for YkV1. Mutations in the GDD RdRp motif, one of the two identifiable functional motifs in the YkV1 polyprotein, abolished its replication competency. Mutations were also introduced in the conserved 2A-like peptide motif, hypothesized to cleave the YkV1 polyprotein cotranslationally. Interestingly, the replication proficiency of YkV1 mutants in the host fungus agreed with the cleavage activity of the 2A-like peptide tested using a baculovirus expression system. Cesium chloride equilibrium density gradient centrifugation allowed for the separation of particles, with a subset of YnV1 capsids solely packaging YkV1 dsRNA and RdRp. These results provide proof of concept that a capsidless positive-sense ssRNA [(1)ssRNA] virus is hosted by an unrelated dsRNA virus. [ABSTRACT FROM AUTHOR]

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.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 2-hexyl, 5-propil resorcinol (HPR). Additionally, PcPCL1606 has biological control against different soil-borne fungal pathogens, including the causal agent of the white root rot of many woody crops and avocado in the Mediterranean area, Rosellinia necatrix. 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 activities in natural conditions and under R. necatrix infection. To test the putative 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 analyzed 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 analyzed, 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.

Rosellinia necatrix, a soilborne fungus, causes white root rot of loquat trees. Toward preventing the disease by enhancing a 45 °C hot water drip irrigation (HWD) treatment, which activates antagonism by soil fungi, we tested three commercial Trichoderma products and found that a product containing T. harzianum T-22 most enhanced the control efficacy. Field evaluation confirmed the enhanced efficacy of HWD with the product to cure the disease. [ABSTRACT FROM AUTHOR]

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 2-hexyl, 5-propil resorcinol (HPR). Additionally, PcPCL1606 has biological control against different soil-borne fungal pathogens, including the causal agent of the white root rot of many woody crops and avocado in the Mediterranean area, Rosellinia necatrix. 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 activities in natural conditions and under R. necatrix infection. To test the putative 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 analyzed 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 analyzed, and PcPCL1606 has a key role in suppressiveness induction; in addition, this phenotype was strongly dependent on the production of HPR. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

• Bacillus velezensis strain S41L highly inhibits four Rosellinia necatrix strains. • S41L severely damages pyriform swellings and the mycelial morphology of R. necatrix. • S41L is capable of IAA and siderophore production, and phosphate solubilization. • B. velezensis strain S41L harbors antibiotic biosynthesis genes. • B. velezensis strain S41L has antifungal and plant growth-promoting properties. White root rot in woody plants is mainly caused by the necrotrophic phytopathogenic fungus Rosellinia necatrix. In this study, we isolate antagonistic bacteria from rhizospheric soil from asymptomatic pear trees infested with white root rot. Furthermore, we isolated 40 bacterial strains and screened them for antagonistic activity against R. necatrix. Then, we selected antagonists that retarded pathogen growth for >30 days. Finally, the strain that exhibited >80 % inhibitory activity against all R. necatrix strains (Korean Agricultural Culture Collection 40446, 40445, 40447, and 40168), demonstrating its effectiveness in controlling this pathogen prevalent in Korean pear and apple orchards, was selected for further experiments. Strain S41L (OP349111) showed the most potent growth-inhibitory activity against the tested phytopathogens. Microbiological and 16S rRNA gene sequencing analyses revealed that the bacterial isolate belonged to the Bacillus genus and had 100 % nucleotide sequence similarity with Bacillus velezensis in GenBank. This strain also harbored antibiotic biosynthesis genes and possessed plant growth-promoting traits such as phosphate solubilization, indole-3-acetic acid, and siderophore production. [ABSTRACT FROM AUTHOR]

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.

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. [ABSTRACT FROM AUTHOR]

The rhizobacterium Pseudomonas pseudoalcaligenes AVO110, isolated by enrichment of competitive avocado root tip colonizers, controls avocado white root rot disease caused by Rosellinia necatrix. Here, we applied signature-tagged mutagenesis (STM) during the growth and survival of AVO110 in fungal exudate-containing medium with the goal of identifying molecular mechanisms linked to the interaction of this bacterium with R. necatrix. A total of 26 STM mutants out-competed by the parental strain in fungal exudate, but not in rich medium, were selected and named growth-attenuated mutants (GAM). Twenty-one genes were identified as required for this bacterial-fungal interaction, including membrane transporters, transcriptional regulators and genes related to the metabolism of hydrocarbons, amino acids, fatty acids and aromatic compounds. The bacterial traits identified here that are involved in the colonization of fungal hyphae include proteins involved in membrane maintenance (a dynamin-like protein and ColS) or cyclic-di-GMP signaling and chemotaxis. In addition, genes encoding a DNA helicase (recB) and a regulator of alginate production (algQ) were identified as required for efficient colonization of the avocado rhizosphere. [ABSTRACT FROM AUTHOR]

The white rot root disease caused by Rosellinia necatrix is a major concern for avocado cultivation in Spain. Healthy escapes of avocado trees surrounded by diseased trees prompted us to hypothesize the presence of hypovirulent R. necatrix due to mycovirus infections. Recently, we reported the presence of another fungal species, Entoleuca sp., belonging to the Xylariaceae, that was also found in healthy avocado trees and frequently co-infecting the same roots than R. necatrix. We investigated the presence of mycoviruses that might explain the hypovirulence. For that, we performed deep sequencing of dsRNAs from two isolates of Entoleuca sp. that revealed the simultaneous infection of several mycoviruses, not described previously. In this work, we report a new member of the Hypoviridae, tentatively named Entoleuca hypovirus 1 (EnHV1). The complete genome sequence was obtained for two EnHV1 strains, which lengths resulted to be 14,958 and 14,984 nt, respectively, excluding the poly(A) tails. The genome shows two ORFs separated by a 32-nt inter-ORF, and both 5′- and 3′-UTRs longer than any other hypovirus reported to date. The analysis of virus-derived siRNA populations obtained from Entoleuca sp. demonstrated antiviral silencing activity in this fungus. We screened a collection of Entoleuca sp. and R. necatrix isolates and found that EnHV1 was present in both fungal species. A genetic population analysis of EnHV1 strains revealed the presence of two main clades, each of them including members from both Entoleuca sp. and R. necatrix, which suggests intra- and interspecific virus transmission in the field. Several attempts failed to cure Entoleuca sp. from EnHV1. However, all Entoleuca sp. isolates collected from avocado, whether harboring the virus or not, showed hypovirulence. Conversely, all R. necatrix isolates were pathogenic to that crop, regardless of being infected by EnHV1.

The antifungal protein (AFP) produced by Aspergillus giganteus, encoded by the afp gene, has been used to confer resistance against a broad range of fungal pathogens in several crops. In this research, transgenic olive plants expressing the afp gene under the control of the constitutive promoter CaMV35S were generated and their disease response against two root infecting fungal pathogens, Verticillium dahliae and Rosellinia necatrix, was evaluated. Embryogenic cultures derived from a mature zygotic embryo of cv. ‘Picual’ were used for A. tumefaciens transformation. Five independent transgenic lines were obtained, showing a variable level of afp expression in leaves and roots. None of these transgenic lines showed enhanced resistance to Verticillium wilt. However, some of the lines displayed a degree of incomplete resistance to white root rot caused by R. necatrix compared with disease reaction of non-transformed plants or transgenic plants expressing only the GUS gene. The level of resistance to this pathogen correlated with that of the afp expression in root and leaves. Our results indicate that the afp gene can be useful for enhanced partial resistance to R. necatrix in olive, but this gene does not protect against V. dahliae.

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. [ABSTRACT FROM AUTHOR]

The antifungal protein (AFP) produced by Aspergillus giganteus, encoded by the afp gene, has been used to confer resistance against a broad range of fungal pathogens in several crops. In this research, transgenic olive plants expressing the afp gene under the control of the constitutive promoter CaMV35S were generated and their disease response against two root infecting fungal pathogens, Verticillium dahliae and Rosellinia necatrix, was evaluated. Embryogenic cultures derived from a mature zygotic embryo of cv. 'Picual' were used for A. tumefaciens transformation. Five independent transgenic lines were obtained, showing a variable level of afp expression in leaves and roots. None of these transgenic lines showed enhanced resistance to Verticillium wilt. However, some of the lines displayed a degree of incomplete resistance to white root rot caused by R. necatrix compared with disease reaction of non-transformed plants or transgenic plants expressing only the GUS gene. The level of resistance to this pathogen correlated with that of the afp expression in root and leaves. Our results indicate that the afp gene can be useful for enhanced partial resistance to R. necatrix in olive, but this gene does not protect against V. dahliae. [ABSTRACT FROM AUTHOR]

The white rot root disease caused by Rosellinia necatrix is a major concern for avocado cultivation in Spain. Healthy escapes of avocado trees surrounded by diseased trees prompted us to hypothesize the presence of hypovirulent R. necatrix due to mycovirus infections. Recently, we reported the presence of another fungal species, Entoleuca sp., belonging to the Xylariaceae, that was also found in healthy avocado trees and frequently co-infecting the same roots than R. necatrix. We investigated the presence of mycoviruses that might explain the hypovirulence. For that, we performed deep sequencing of dsRNAs from two isolates of Entoleuca sp. that revealed the simultaneous infection of several mycoviruses, not described previously. In this work, we report a new member of the Hypoviridae, tentatively named Entoleuca hypovirus 1 (EnHV1). The complete genome sequence was obtained for two EnHV1 strains, which lengths resulted to be 14,958 and 14,984 nt, respectively, excluding the poly(A) tails. The genome shows two ORFs separated by a 32-nt inter-ORF, and both 50- and 30-UTRs longer than any other hypovirus reported to date. The analysis of virus-derived siRNA populations obtained from Entoleuca sp. demonstrated antiviral silencing activity in this fungus. We screened a collection of Entoleuca sp. and R. necatrix isolates and found that EnHV1 was present in both fungal species. A genetic population analysis of EnHV1 strains revealed the presence of two main clades, each of them including members from both Entoleuca sp. and R. necatrix, which suggests intra- and interspecific virus transmission in the field. Several attempts failed to cure Entoleuca sp. from EnHV1. However, all Entoleuca sp. isolates collected from avocado, whether harboring the virus or not, showed hypovirulence. Conversely, all R. necatrix isolates were pathogenic to that crop, regardless of being infected by EnHV1. [ABSTRACT FROM AUTHOR]

Avocado white root rot is caused primarily by Rosellinia necatrix Prill. In this study, we buried bait twigs around avocado escape trees to recover non-pathogenic soil-borne fungi. First, we selected forty isolates: 19 were identified as R. necatrix and 21 as Entoleuca sp. The next step was to conduct pathogenicity tests on lupin and avocado plants, which showed that R. necatrix isolates were pathogenic in both types of plants, while Entoleuca sp. isolates were not. The optimal growth temperature in vitro for the Entoleuca sp. isolates was 30 °C. Dianthus caryophyllus was found to be a host to this fungus, while Olea europaea , Lupinus luteus , Asparagus officinalis and Fragaria ananassa were not. Next, Entoleuca sp. was artificially inoculated into avocado plants and recovered from the roots up to two years later. In this way, we tested the biocontrol of the disease using Entoleuca sp. in avocado plants artificially inoculated with R. necatrix. Most of Entouleca isolates (86%) controlled the disease, and we therefore concluded that they were effective biocontrol agents. [ABSTRACT FROM AUTHOR]

Rosellinia necatrix: causing root rot disease is a very destructive pathogen of woody plants and is responsible for yield losses to a large number of fruit trees. The genetic analysis of this pathogen has not been picked up because of difficulty in generating mutations in Rosellinia necatrix for many reasons. A number of methods have been proposed for inducing mutations in Rosellinia necatrix but none of them proved worth because of very low transformation efficiencies. Here, we propose an efficient method for Rosellinia necatrix protoplast production, where protoplasts in the tune of 10 per ml can be easily generated. We also propose a restriction enzyme-mediated integration (REMI)-based methods for efficient transformation of Rosellinia necatrix. In the present study, an approximate of 800 transformants was obtained from 5 μg of linearized plasmid. Out of 47 single spored transformants analyzed, only 33 showed hygromycin gene amplification using PCR and only 19 transformants showed single gene integration in southern hybridization, which accounted for single gene integration percentage of 42%, highest amongst all the previous reports on Rosellinia necatrix transformations. Some of the transformants studied for pathogenicity phenotype also showed a marked reduction in pathogenicity. Thus, in the present investigation, 42% single gene integrations among the transformed colonies can be considered as excellent transformation efficiency. [ABSTRACT FROM AUTHOR]

The biocontrol rhizobacterium Pseudomonas chlororaphis PCL1606 has the ability to protect avocado plants against white root rot produced by the phytopathogenic fungus Rosellinia necatrix. Moreover, PCL1606 displayed direct interactions with avocado roots and the pathogenic fungus. Thus, nonmotile (flgK mutant) and non-chemotactic (cheA mutant) derivatives of PCL1606 were constructed to emphasize the importance of motility and chemotaxis in the biological behaviour of PCL1606 during the biocontrol interaction. Plate chemotaxis assay showed that PCL1606 was attracted to the single compounds tested, such as glucose, glutamate, succinate, aspartate and malate, but no chemotaxis was observed to avocado or R. necatrix exudates. Using the more sensitive capillary assay, it was reported that smaller concentrations (1 mM) of single compounds elicited high chemotactic responses, and strong attraction was confirmed to avocado and R. necatrix exudates. Finally, biocontrol experiments revealed that the cheA and fglK derivative mutants reduced root protection against R. necatrix, suggesting an important role for these biological traits in biocontrol by P. chlororaphis PCL1606. [ABSTRACT FROM AUTHOR]

The family Partitiviridae comprises of five genera with bi-segmented dsRNA genomes that accommodate members infecting plants, fungi or protists. All partitiviruses with only a few exceptions cause asymptomatic infections. We report the characterization of a novel betapartitivirus termed Rosellinia necatrix partitivirus 6 (RnPV6) from a field isolate of a plant pathogenic fungus, white root rot fungus. RnPV6 has typical partitivirus features: dsRNA1 and dsRNA2 are 2462 and 2499 bps in length encoding RNA-dependent RNA polymerase and capsid protein. Purified particles are spherical with a diameter of 30 nm. Taking advantage of infectivity as virions, RnPV6 was introduced into a model filamentous fungal host, chestnut blight fungus to investigate virus/host interactions. Unlike other partitiviruses tested previously, RnPV6 induced profound phenotypic alterations with symptoms characterized by a reduced growth rate and enhanced pigmentation and was tolerant to host RNA silencing. In addition, a variety of defective RNAs derived from dsRNA1 appear after virion transfection. These sub-viral RNAs were shown to interfere with RnPV6 replication, at least for that of cognate segment dsRNA1. Presence of these sub-viral elements resulted in reduced symptom expression by RnPV6, suggesting their nature as defective-interfering RNAs. The features of RnPV6 are similar to but distinct from those of a previously reported alphapartitivirus, Rosellinia necatrix partitivirus 2 that is susceptible to RNA silencing. [ABSTRACT FROM AUTHOR]

A new virus termed Rosellinia necatrix megabirnavirus 2 (RnMBV2) was molecularly and biologically characterized. RnMBV2 was originally harbored in isolate W8 of R. necatrix co-infected with the previously reported virus Rosellinia necatrix partitivirus 1 (RnPV1). RnMBV2 has molecular features similar and different from precedent megabirnaviruses, Rosellinia necatrix megabirnavirus 1 (RnMBV1) and Sclerotinia sclerotiorum megabirnavirus 1 (SsMBV1). The two genomic segments of RnMBV2 (9.0-kbp dsRNA1 and 8.0-kbp dsRNA2) each possess two open reading frames (ORF1 and 2 on dsRNA1 and ORF3 and 4 on dsRNA2), with a well conserved 5′-long untranslated region (UTR) of 1.7–1.8 kb between the segments, and relatively short 3′-UTR. The RnMBV2 dsRNA1-coded capsid protein (CP) and RNA-dependent RNA polymerase (RdRp) show higher sequence identity to those of SsMBV1 than to those of RnMBV1, whereas the RnMBV2 ORF3-coded protein is more closely related to the counterpart of RnMBV1. No significant amino acid sequence similarity was detected among ORF4-coded sequences of the three megabirnaviruses. Virion transfection and co-culturing allowed for single and double infection of mycelial incompatible isolates W37 and W97 by RnMBV2 and/or RnPV1. Their comparative analyses showed RnMBV2 to be able to confer hypovirulence with the aid of a co-infecting RnPV1, while the individual viruses exhibited asymptomatic infections. Interestingly, RnPV1 accumulation appeared to be increased in co-infected fungal strain with two segments of RnMBV2 relative to singly infected fungal strains. Furthermore, the dispensability of RnMBV2 dsRNA2 was demonstrated to be similar to that of the other two megabirnaviruses. [ABSTRACT FROM AUTHOR]