The soil-borne white root rot pathogen Rosellinia necatrix expresses antimicrobial proteins during host colonization.

Rosellinia necatrix is a prevalent soil-borne plant-pathogenic fungus that is the causal agent of white root rot disease in a broad range of host plants. The limited availability of genomic resources for R. necatrix has complicated a thorough understanding of its infection biology. Here, we sequenced nine R. necatrix strains with Oxford Nanopore sequencing technology, and with DNA proximity ligation we generated a gapless assembly of one of the genomes into ten chromosomes. Whereas many filamentous pathogens display a so-called two-speed genome with more dynamic and more conserved compartments, the R. necatrix genome does not display such genome compartmentalization. It has recently been proposed that fungal plant pathogens may employ effectors with antimicrobial activity to manipulate the host microbiota to promote infection. In the predicted secretome of R. necatrix, 26 putative antimicrobial effector proteins were identified, nine of which are expressed during plant colonization. Two of the candidates were tested, both of which were found to possess selective antimicrobial activity. Intriguingly, some of the inhibited bacteria are antagonists of R. necatrix growth in vitro and can alleviate R. necatrix infection on cotton plants. Collectively, our data show that R. necatrix encodes antimicrobials that are expressed during host colonization and that may contribute to modulation of host-associated microbiota to stimulate disease development.

Susceptibility of Novel Promising Citrus Rootstocks to White Root Rot.

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.

A moderate level of hypovirulence conferred by a hypovirus in the avocado white root rot fungus, Rosellinia necatrix.

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.

The Strawberry FaWRKY1 Transcription Factor Negatively Regulates Resistance to Colletotrichum acutatum in Fruit Upon Infection.

Strawberry (Fragaria ×ananassa) is a major food crop worldwide, due to the flavor, aroma and health benefits of the fruit, but its productivity and quality are seriously limited by a large variety of phytopathogens, including Colletotrichum spp. So far, key factors regulating strawberry immune response remain unknown. The FaWRKY1 gene has been previously proposed as an important element mediating defense responses in strawberry to Colletotrichum acutatum. To get further insight into the functional role that FaWRKY1 plays in the defense mechanism, Agrobacterium-mediated transient transformation was used both to silence and overexpress the FaWRKY1 gene in strawberry fruits (Fragaria ×ananassa cv. Primoris), which were later analyzed upon C. acutatum inoculation. Susceptibility tests were performed after pathogen infection comparing the severity of disease between the two agroinfiltrated opposite halves of the same fruit, one half bearing a construct either for FaWRKY1 overexpression or RNAi-mediated silencing and the other half bearing the empty vector, as control. The severity of tissue damage was monitored and found to be visibly reduced at five days after pathogen inoculation in the fruit half where FaWRKY1 was transiently silenced compared to that of the opposite control half and statistical analysis corroborated a significant reduction in disease susceptibility. Contrarily, a similar level of susceptibility was found when FaWRKY1 overexpression and control fruit samples, was compared. These results unravel a negative regulatory role of FaWRKY1 in resistance to the phytopathogenic fungus C. acutatum in strawberry fruit and contrast with the previous role described for this gene in Arabidopsis as positive regulator of resistance against the bacteria Pseudomonas syringae. Based on previous results, a tentative working model for WRKY75 like genes after pathogen infection is proposed and the expression pattern of potential downstream FaWRKY1 target genes was also analyzed in strawberry fruit upon C. acutatum infection. Our results highlight that FaWRKY1 might display different function according to species, plant tissue and/or type of pathogen and underline the intricate FaWRKY1 responsive defense regulatory mechanism taking place in strawberry against this important crop pathogen.