Evaluation of Fungal Volatile Organic Compounds for Control the Plant Parasitic Nematode Meloidogyne incognita.

Plant parasitic nematodes are a serious threat to crop production worldwide and their control is extremely challenging. Fungal volatile organic compounds (VOCs) provide an ecofriendly alternative to synthetic nematicides, many of which have been withdrawn due to the risks they pose to humans and the environment. This study investigated the biocidal properties of two fungal VOCs, 1-Octen-3-ol and 3-Octanone, against the widespread root-knot nematode Meloidogyne incognita. Both VOCs proved to be highly toxic to the infective second-stage juveniles (J2) and inhibited hatching. Toxicity was dependent on the dose and period of exposure. The LD50 of 1-Octen-3-ol and 3-Octanone was 3.2 and 4.6 µL, respectively. The LT50 of 1-Octen-3-ol and 3-Octanone was 71.2 and 147.1 min, respectively. Both VOCs were highly toxic but 1-Octen-3-ol was more effective than 3-Octanone. Exposure of M. incognita egg-masses for 48 h at two doses (0.8 and 3.2 µL) of these VOCs showed that 1-Octen-3-ol had significantly greater nematicidal activity (100%) than 3-Octanone (14.7%) and the nematicide metham sodium (6.1%). High levels of reactive oxygen species detected in J2 exposed to 1-Octen-3-ol and 3-Octanone suggest oxidative stress was one factor contributing to mortality and needs to be investigated further.

Evaluation of Metarhizium brunneum- and Metarhizium-Derived VOCs as Dual-Active Biostimulants and Pest Repellents in a Wireworm-Infested Potato Field.

Wireworm, the larval stages of click beetles, are a serious pest of tubers, brassicas and other important commercial crops throughout the northern hemisphere. No effective control agent has been developed specifically for them, and many of the pesticides marketed as having secondary application against them have been withdrawn from EU and Asian markets. Metarhizium brunneum, an effective entomopathogenic fungus, and its derived volatile metabolites are known to be effective plant biostimulants and plant protectants, although field efficacy has yet to be validated. Field validation of a combined M. brunneum and derived VOC treatments was conducted in Wales, UK, to assess the effects of each as a wireworm control agent and biostimulant. Plots were treated with Tri-Soil (Trichoderma atroviridae), M. brunneum, 1-octen-3-ol or 3-octanone, or combinations thereof. Treatments were applied subsurface during potato seeding (n = 52), and potatoes were harvested at the end of the growing season. Each potato was weighed individually and scored for levels of wireworm damage. Applications of both the VOCs and the M. brunneum individually were found to significantly decrease wireworm burden (p < 0.001). Combinations of M. brunneum and 3-octanone were also found to significantly decrease wireworm damage (p < 0.001), while no effect on yield was reported, resulting in an increased saleable mass over controls (p < 0.001). Herein, we present a novel 'stimulate and deter' wireworm control strategy that can be used to significantly enhance saleable potato yields and control wireworm populations, even under high pest pressure densities.

Metarhizium brunneum (Hypocreales: Clavicipitaceae) and Its Derived Volatile Organic Compounds as Biostimulants of Commercially Valuable Angiosperms and Gymnosperms.

Metarhizium brunneum is a highly effective entomopathogenic fungus that also functions as a plant biostimulant. It can act as both an endophyte and rhizosphere colonizer; however, the mechanisms driving biostimulation are multifactorial. In this work, oilseed rape (Brassica napus) seeds were grown in composts treated with different concentrations of M. brunneum strains ARSEF 4556 or V275, or the M. brunneum-derived volatile organic compounds 1-octen-3-ol and 3-octanone. Biostimulation efficacy was found to be strongly dose dependent. Concentrations of 1 × 106 conidia g-1 compost were found to be most effective for the M. brunneum, whereas dosages of 1 µL 100 g-1 compost were found to be efficacious for the volatiles. These optimized doses were assessed individually and in combined formulations with a hydrogel against oilseed rape (Brassica napus), sitka spruce (Picea sitchensis), maize (Zea mays) and strawberry (Fragaria annanassa). Both volatile compounds were highly effective biostimulants and were found to increase in biostimulatory efficiency when combined with M. brunneum conidia. Hydrogels were not found to interact with the growth process and may offer avenues for novel formulation technologies. This study demonstrates that Metarhizium-derived volatile organic compounds are actively involved in plant growth promotion and have potential for use in novel formulations to increase the growth of a wide range of commercially relevant crops.

Volatiles of the entomopathogenic fungus, Metarhizium brunneum, attract and kill plant parasitic nematodes.

Root knot nematodes (RKNs) cause significant crop losses. Although RKNs and entomopathogenic fungi, such as Metarhizium brunneum, are associated with plant roots, very little is known about the interactions between these two organisms. This study showed that conidia and VOCs of Me. brunneum influenced the behaviour of M. hapla. The response was dependent on the fungal strain, VOC, concentration of both VOC and conidia, and time. Tomatoes planted in soil treated with the highest doses of conidia usually had a higher number of nematodes than untreated control plants. This was particularly obvious for Me. brunneum strain ARSEF 4556, 7 and 14-days post-treatment. The VOCs, 1-octen-3-ol and 3-octanone, lured M. hapla to plants when used at low doses and repelled them at high doses. In Petri dish assays. the VOCs 1-octen-3-ol and 3-octanone, caused 100% mortality of M. hapla at the highest dose tested (20 µl). Very few live M. hapla were recovered from soil treated with the VOC 1-octen-3-ol, especially at the highest doses tested.

'Candidatus Phytoplasma convolvuli', a new phytoplasma taxon associated with bindweed yellows in four European countries.

Plants of Convolvulus arvensis exhibiting symptoms of undersized leaves, shoot proliferation and yellowing, collectively defined as bindweed yellows, were sampled in different regions of Europe and assessed for phytoplasma infection by PCR amplification using phytoplasma universal rRNA operon primer pairs. Positive results were obtained for all diseased plants. RFLP analysis of amplicons comprising the16S rRNA gene alone or the16S rRNA gene and 16-23S intergenic spacer region indicated that the detected phytoplasmas were distinguishable from all other previously described rRNA gene sequences. Analysis of 16S rRNA gene sequences derived from seven selected phytoplasma strains (BY-S57/11, BY-S62/11, BY-I1015, BY-I1016, BY-BH1, BY-BH2 and BY-G) showed that they were nearly identical (99.9-100% gene sequence similarity) but shared less than 97.5% similarity with comparable sequences of other phytoplasmas. Thus, BY phytoplasmas represent a new taxon whose closest relatives are stolbur phytoplasma strains and 'Candidatus Phytoplasma fragariae' with which they share 97.2% and 97.1% 16S rRNA gene sequence similarity, respectively. Phylogenetic analysis of 16S rRNA gene sequences confirmed that bindweed yellows phytoplasma strains collectively represent a distinct lineage within the phytoplasma clade and share a common ancestor with previously published or proposed 'Candidatus Phytoplasma' taxa within a major branch including aster yellows and stolbur phytoplasmas. On the basis of unique 16S rRNA gene sequences and biological properties that include a single host plant species and a geographical distribution limited to parts of Europe, the bindweed yellows (BY) phytoplasmas represent a coherent but discrete taxon, 'Candidatus Phytoplasma convolvuli', with strain BY-S57/11 (GenBank accession no. JN833705) as the reference strain.

'Kwanzan Stunting' syndrome: detection and molecular characterization of an Italian isolate of Little cherry virus 1.

Evident stunting was observed for the first time on Prunus serrulata 'Kwanzan' indicator trees in Southern Italy during the indexing of two sour cherry accessions from cultivars 'Marasca di Verona' and 'Spanska'. Bud break and shooting were delayed and the developing leaves remained small. During the third year many Kwanzan plants died, regardless of the indexed cultivar. Electrophoretic analysis showed the presence of dsRNA pattern in extracts of stunted Kwanzan with a similar size to that of viruses of the family Closteroviridae. An identical pattern of more abundant dsRNA bands was obtained from GF305 seedlings grafted with the same sour cherry accessions. Observations by electron microscopy revealed the presence of long flexuous virus particles in both indicators (Kwanzan and GF305), characteristic of closteroviruses. Subsequent cloning work, starting from the dsRNA extracts of cultivar Marasca di Verona grafted on GF305 indicator, yielded 7 different clones, all showing high identity to the Little cherry virus 1 genome. Full sequencing of this virus isolate (ITMAR) was then done resulting in a complete genome composed of 16,936nt. Primers designed on the obtained sequences for RT-PCR detection confirmed the presence of Little cherry virus 1 in Kwanzan and GF305 trees, inoculated with both sour cherry cultivars. Phylogenetic analysis of the minor coat protein grouped virus isolates into two clusters: one including Italian isolates of sweet cherry, Japanese plum, peach and almond, together with German sweet cherry UW1 isolate, and a second one containing the Italian isolates of sour cherry (ITMAR and ITSPA), that were found associated with strong symptoms of 'Kwanzan Stunting'.

Simultaneous detection and identification of four pome fruit viruses by one-tube pentaplex RT-PCR.

A pentaplex reverse-transcription polymerase chain reaction (Pentaplex RT-PCR) in a single tube was developed for the simultaneous detection of the pome fruit viruses: Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV), Apple chlorotic leaf spot virus (ACLSV) and Apple mosaic virus (ApMV). This is the first report of the simultaneous detection of all four viruses and host mRNA as an internal specific control. Pentaplex RT-PCR was applied successfully throughout the year, using different plant organs (leaves or dormant buds). The sensitivity of detection by monoplex- and pentaplex RT-PCR assays was comparable. Different combinations of mixed infections of viruses were identified in samples of infected apple and pear trees from different geographical regions. The pentaplex RT-PCR assay developed was sensitive, simple, rapid, and reliable for simultaneous detection of the four viruses in extracts of leaves or dormant buds.

The molecular characterization of 16 new sequence variants of Hop stunt viroid reveals the existence of invariable regions and a conserved hammerhead-like structure on the viroid molecule.

At present isolates of Hop stunt viroid (HSVd) are divided into five groups: three major groups (plum-type, hop-type and citrus-type) each containing isolates from only a limited number of isolation hosts and two minor groups that were presumed to derive from recombination events between members of the main groups. In this work we present the characterization of 16 new sequence variants of HSVd obtained from four Mediterranean countries (Cyprus, Greece, Morocco and Turkey) where this viroid had not previously been described. Molecular variability comparisons considering the totality of the sequence variants characterized so far revealed that most of the variability is found in the pathogenic and variable domains of the viroid molecule whereas both the terminal right (T(R)) and left (T(L)) domains are regions of low or no variability, respectively, suggesting the existence of constraints limiting the heterogeneity of the sequence variants. Phylogenetic analyses revealed that sequence variants belonging to the two minor recombinant subgroups are more frequent than previously thought. When the cruciform structure alternative to the typical rod-like conformation was considered it was observed that the upper part of this structure (hairpin I) was strictly conserved whereas in the lower part a reduced variability was found. The existence of a covariation in this lower part was notable. Interestingly, a hammerhead-like sequence was found within the T(R) domain of HSVd and it was strictly conserved in all the sequence variants. The evolutionary implications of the presence of this motif on the HSVd are discussed.