Transcriptome analysis of the fungal pathogen Rosellinia necatrix during infection of a susceptible avocado rootstock identifies potential mechanisms of pathogenesis.

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.

Pharmacological modulation of LMNA SRSF1-dependent splicing abrogates diet-induced obesity in mice.

Bakground/Objectives:Intense drug discovery efforts in the metabolic field highlight the need for novel strategies for the treatment of obesity. Alternative splicing (AS) and/or polyadenylation enable the LMNA gene to express distinct protein isoforms that exert opposing effects on energy metabolism and lifespan. Here we aimed to use the splicing factor SRSF1 that contribute to the production of these different isoforms as a target to uncover new anti-obesity drug. SUBJECTS/METHODS: Small molecules modulating SR protein activity and splicing were tested for their abilities to interact with SRSF1 and to modulate LMNA (AS). Using an LMNA luciferase reporter we selected molecules that were tested in diet-induced obese (DIO) mice. Transcriptomic analyses were performed in the white adipose tissues from untreated and treated DIO mice and mice fed a chow diet. RESULTS: We identified a small molecule that specifically interacted with the RS domain of SRSF1. ABX300 abolished DIO in mice, leading to restoration of adipose tissue homeostasis. In contrast, ABX300 had no effect on mice fed a standard chow diet. A global transcriptomic analysis revealed similar profiles of white adipose tissue from DIO mice treated with ABX300 and from untreated mice fed a chow diet. Mice treated with ABX300 exhibited an increase in O2 consumption and a switch in fuel preference toward lipids. CONCLUSIONS: Targeting SRSF1 with ABX300 compensates for changes in RNA biogenesis induced by fat accumulation and consequently represents a novel unexplored approach for the treatment of obesity.

Use of Poultry Manure Combined with Soil Solarization as a Control Method for Meloidogyne incognita in Carnation.

The effectiveness of a combination of soil solarization and poultry manure (raw or pelletized) amendments for the control of root-knot nematode (Meloidogyne incognita) was tested in carnation (Dianthus caryophyllus) crops grown in in-ground beds under plastic-covered greenhouse conditions in southern Spain. Our trials demonstrated that soil solarization alone did not provide sufficient control of root-knot nematode, because the carnation growing season in this region only partly coincides with the most effective period for solarization, resulting in an insufficient duration of treatment during a key period for effectiveness. Chemical fumigation with 1,3-dichloropropene + chloropicrin prior to planting was effective in reducing nematode population densities in soil. Its effects spanned 9 months after planting, resulting in acceptable crop yields. In comparison, the combination of soil solarization and raw or pelletized poultry manure was slightly less effective than chemical fumigation for control of this pathogen but crop yields after 9 months were similar. However, the higher root gall indices observed after 9 months, in comparison with chemically fumigated plots, indicated the need for a reapplication of the organic manure treatment at the start of each successive growing season.

GFP sheds light on the infection process of avocado roots by Rosellinia necatrix.

In order to monitor Rosellinia necatrix infection of avocado roots, we generated a plasmid vector (pCPXHY1eGFP) constitutively expressing EGFP and developed a protoplast transformation protocol. Using this protocol, four R. necatrix isolates were efficiently transformed and were shown to stably express EGFP homogeneously while not having any observable effect on pathogenicity. Confocal laser scanning microscopy (CLSM) images of avocado roots infected with the highly virulent isolate CH53-GFP demonstrated that fungal penetration of avocado roots occurs simultaneously at several random sites, but it occurs preferentially in the crown region as well as throughout the lenticels and in the junctions between epidermal cells. Not only were R. necatrix hyphae observed invading the epidermal and cortical root cells, but they were also able to penetrate the primary and secondary xylem. Scanning electron microscopy (SEM) images allowed detailed visualisation of the hyphal network generated by invasion of R. necatrix through the epidermal, cortical and vascular cells, including hyphal anastomosis and branching points. To our knowledge, this is the first report describing the construction of GFP-tagged strains belonging to the genus Rosellinia for monitoring white root rot using CLSM and SEM.

First Report of Phytophthora cactorum Causing Fruit Rot on Avocado in Spain.

The area of avocado (Persea americana Mill.) orchards in southern Spain has increased recently and is currently at 8,063 ha. Avocado production in this part of Spain was 72,581 t during 2003. During February 2004, apical necrosis was observed on avocado fruits (cv. Hass) in one orchard in Vélez-Málaga, Málaga Province, southern Spain. Dark brown lesions and necrotic flecking of the flesh also were observed on fruits. Isolations from the skin of the fruit previously washed with tap water and disinfested with 20% sodium hypochlorite on potato dextrose agar (PDA) consistently resulted in mycelial colonies. Sporangia produced on V8 juice by successive washing of mycelia with saline solution (1) measured 31 to 37.2 (33.3) × 21.7 to 28.8 (24.2) µm in size. The pathogen was identified as Phytophthora cactorum on the basis of morphological structures (mycelia, sporangia, chlamydospores, and oospores) formed when grown on V8 juice and PDA (2). To confirm pathogenicity, a mycelial suspension was obtained by blending mycelia grown for 1 week on PDA in 200 ml of sterile water. Three healthy avocado fruits were inoculated with the suspension by injection; three other fruits were inoculated by placing a drop of suspension on the unbroken skin of the fruit. The same number of fruit was inoculated as controls using sterile water instead of mycelial suspension. The inoculated fruits were incubated for 5 days in a moist chamber at 24°C in darkness. Spots appeared on all fruits for both inoculation methods, and the pathogen was isolated and identified as P. cactorum. No symptoms appeared on the control fruits. To our knowledge, this is the first report of P. cactorum causing fruit rot on avocado in Spain. References: (1) D. Chen and G. A. Zentmeyer. Mycologia 62:397, 1970. (2) G. M. Waterhouse and J. M. Waterston. No. 111 in: Descriptions of Pathogenic Fungi and Bacteria. CMI, Kew, Surrey, UK, 1966.

Soil Solarization in Established Avocado Trees for Control of Dematophora necatrix.

Four field experiments on the control of Dematophora necatrix in avocado orchards affected by white root rot were conducted in the Mediterranean coastal area of southern Spain during 1991 to 1994. In the unshaded locations of solarized plots, the maximal temperatures were 35 to 42°C, depending upon the year and soil depth (15 to 60 cm). Temperature increases attributable to soil solarization ranged between 4 and 8°C in unshaded areas, whereas for shaded areas they were approximately 4°C. Inoculum recovery was decreased in root samples buried at 15 to 30 cm in unshaded locations of both solarized and unsolarized plots after 3 to 5 weeks, whereas 4 to 8 weeks of solarization were required for the elimination of the pathogen buried at depths of 45 to 60 cm. In contrast, inoculum recovery ranged from 30 to 60% for samples in shaded locations of unsolarized plots. D. necatrix was not recovered from roots of infected trees in solarized plots sampled 9 months after solarization, whereas recovery from roots in unsolarized plots was similar to levels before solarization. Soil solarization in established orchards was successful in reducing viability of inoculum buried in soil and eliminated inoculum in infected roots of live trees.