Mycoviruses in Biological Control: From Basic Research to Field Implementation.

Mycoviruses from plant pathogens can induce hypovirulence (reduced virulence) in their host fungi and have gained considerable attention as potential biocontrol tools. An increasing number of mycoviruses that induce fungal hypovirulence, from a wide variety of taxonomic groups, are currently being reported. Successful application of these viruses in disease management is greatly dependent on their ability to spread in the natural populations of the pathogen. Mycoviruses generally lack extracellular routes of transmission. Hyphal anastomosis is the main route of horizontal mycovirus transmission to other isolates, and conidia of vertical transmission to the progeny. Transmission efficiencies are influenced by both the fungal host and the infecting virus. Interestingly, artificial transfection methods have shown that potential biocontrol mycoviruses often have the ability to infect a variety of fungi. This expands their possible use to the control of pathogens others than those where they were identified. Mycovirus research is also focused on gaining insights into their complex molecular biology and the molecular bases of fungus-virus interactions. This knowledge could be exploited to manipulate the mycovirus and/or the host and generate combinations with enhanced properties in biological control. Finally, when exploring the use of mycoviruses in field conditions, the pathogen life style and the characteristics of the disease and crops affected will deeply impact the specific challenges to overcome, and the development of biocontrol formulations and delivery methods.

Study of Phylogenetic Relationships Among Fusarium oxysporum f. sp. dianthi Isolates: Confirmation of Intrarace Diversity and Development of a Practical Tool for Simple Population Analyses.

Fusarium wilt, caused by Fusarium oxysporum f. sp. dianthi, is the most important disease of carnation worldwide. Knowing the diversity of the F. oxysporum f. sp. dianthi population present in a carnation growing area is a key component of preventing dramatic losses in production. Sequence analyses of partial ß-tubulin, translation elongation factor 1α genes, and the full-length ribosomal DNA intergenic spacer (IGS) were conducted to resolve phylogenetic relationships in a wide collection of Spanish F. oxysporum f. sp. dianthi isolates, along with some representatives from Italy. We found that, among the three different gene regions, the IGS sequence was the best choice to resolve phylogenetic relationships among F. oxysporum f. sp. dianthi isolates. The phylogenetic tree generated with the complete IGS region was the only one showing a clear clustering of isolates according to the molecular group (virulence grouping) and the vegetative compatibility group. In order to develop a more practical tool based on a shorter DNA sequence to quickly analyze diversity in F. oxysporum f. sp. dianthi populations, we examined IGS nucleotide alignments and identified a region of approximately 300 bp that accumulates enough "informative" changes to resolve intraspecific relationships and determine pathogenic variants in F. oxysporum f. sp. dianthi. Moreover, the "condensed" alignment of this short IGS region showing only the informative positions revealed the existence of virulence group-discriminating positions. In addition to clarifying the phylogenetic relationships among F. oxysporum f. sp. dianthi isolates of the recently described race groups by using multigene genealogies, we have developed simple tools for the phylogenetic analyses of F. oxysporum f. sp. dianthi populations and the determination of the molecular group of uncharacterized F. oxysporum f. sp. dianthi isolates.

First Report of Verticillium Wilt Caused by Verticillium dahliae on Mango Trees (Mangifera indica) in Southern Spain.

Verticillium wilt, primarily caused by Verticillium dahliae Klebahn and V. albo-atrum Reinke & Berthold, affects a wide range of economically important crops. This disease is an increasing problem in areas where young mango trees are planted on land previously planted in vegetable crops. In 2008, symptoms of Verticillium wilt were observed in mango cvs. Kent and Osteen in the subtropical fruit-producing area of Málaga in southern Spain. In a new mango grove of cv. Kent, previously planted in potatoes and tomatoes, ~20% of 200 1-year-old trees had one-sided branch dieback. In many of these trees the symptoms expanded, leading to decline and eventual death. Cross sections of affected branches revealed brown vascular discoloration. Verticillium was isolated from surface-sterilized segments of symptomatic branches placed on acidic potato dextrose agar (PDA). Plates were incubated at 24°C. After 3 days, slow-growing colonies were transferred to PDA. Verticillium was similarly isolated from symptomatic potato plants grown in a nearby field. Identification of V. dahliae was initially based on morphology and further confirmed by molecular methods. All isolates tested produced microsclerotia, a defining feature that distinguishes V. dahliae from V. albo-atrum. For molecular characterization, V. dahliae specific primers 19 and 22 (1) and universal primers ITS1 and ITS4, which amplify the rRNA internal transcribed spacer (ITS) region (4), were used. Bands of expected size were amplified with both primer combinations. ITS fragments were sequenced and identical to the V. dahliae reference sequence (GenBank AY555948) (3). Pathogenicity assays were conducted with a selected isolate from mango using tomato plants from the susceptible line 'Moneymaker' and the near isogenic 'Motabo' line carrying the Ve gene conferring resistance to race 1 isolates. Five 1-month-old plants (four-leaf stage) were inoculated by root immersion in a suspension of 107 conidia/ml. Five control plants were mock inoculated with distilled water. As a positive control, five plants were inoculated with the previously described race 1 strain Dvd-T5 (2), which induces severe symptoms in susceptible tomato cultivars. Symptoms were scored visually at various time points up to 40 days by a 0 to 5 scale in which 0 = negligible chlorosis or wilting, 1 = chlorosis and wilting and/or curling in individual leaves, 2 = necrosis in leaves, 3 = at least one branch dead, 4 = wilt and/or chlorosis in upper leaves and/or two or more branches dead, and 5 = plant dead or all leaves and most of stem necrotic. The isolate from mango caused typical Verticillium wilt symptoms with a mean disease rating of 3.6 at 40 days postinoculation in both lines. The mean disease rating for Dvd-T5 in Moneymaker 40 days postinoculation was 4.0. V. dahliae was reisolated from symptomatic plants but not from noninoculated controls. To our knowledge, this is the first report of Verticillium wilt on mango in Spain. More problems with Verticillium wilt are expected because of the increasing planting of mango in fields previously dedicated to horticultural crops. References: (1) J. H. Carder et al. Modern Assays for Plant Pathogenic Fungi: Identification, Detection and Quantification. CAB International, Oxford, 1994. (2) K. F. Dobinson et al. Can. J. Plant. Pathol. 18:55, 1996. (3) M. P. Pantou et al. Mycol. Res. 109:889, 2005. (4) T. J. White et al. PCR Protocols: A Guide to Methods and Amplification. Academic Press, San Diego, 1990.

New (and used) approaches to the study of fungal pathogenicity.

The fungi are the most economically important plant pathogens and continue to be the focus of extensive research with a wide variety of methodologies. Enhancements in microscopy techniques have increased our ability to visualize the intimate interaction of fungi and their host plants. Improving methods allow pharmacological inhibition and genetic dissection of the determinants of fungal pathogenicity in a gene-by-gene approach. Identification and analysis of genes differentially transcribed in ways pertinent to pathogenicity continues to be a frequent research approach. Genome-wide analysis is gaining favor in biological research and fungal plant pathogens are no exception. Several industrial research groups are exploring fungal plant pathogenesis based on genomic sequence data and genome-wide mutagenesis. In March 2001 the first publicly available complete genome of a filamentous fungus (Neurospora crassa) was released. N. crassa is of course a saprophyte and there is no complete sequence available for a plant pathogenic fungus in public databases. However, freely accessible entire genome sequences for both plant pathogenic fungi and their hosts are on the horizon. Sequence availability promises to revolutionize the rate at which data relevant to disease processes will be accrued. In this review we describe approaches currently applied to the study of plant pathogenic fungi and explore developments of potential future benefit with existing technologies not yet applied to this group of important organisms.

A homologous and self-replicating system for efficient transformation of Fusarium oxysporum.

A highly efficient transformation system has been developed for Fusarium oxysporum f. sp. lycopersici based on the complementation of a nitrate-reductase mutant with the homologous nit1 gene and on the presence of ARS and telomeric sequences in the vector. Preliminary transformation experiments with the niaD gene from Aspergillus niger generated self-replicating plasmids within the transformed entity that contained extra-fungal DNA. A fragment of the extra DNA was inserted into pUC19 together with the F. oxysporum nit1 gene, resulting in plasmid pFNit-Lam. This allowed the isolation of a new linear plasmid within self-replicative F. oxysporum transformants (pFNit-Lam-TLam, linear). The circular form of this vector yielded 5600 fungal transformants per microgram of DNA. All of the transformants contained autonomous linear plasmids harboring direct repeats of fungal DNA at both ends. The sequence of the 1.2-kb fragment from F. oxysporum responsible for autonomous replication, and maintenance as linear plasmid molecules, has been determined. Comparison analysis with the ARS from different organisms has shown that this fragment contained the commonly identified ARS consensus sequence, 5'A/TTTTATA/GTTTA/T3' and, in addition to this core, ten copies of the ARS-box, 5'TNTA/GAA3'. Adjacent to this presumed ARS, the telomeric hexanucleotide sequence (TTAGGG)n was present in six tandem copies followed by 18 copies of its complementary sequence.

An association between mutagenicity of the Ara test of Salmonella typhimurium and carcinogenicity in rodents for 16 halogenated aliphatic hydrocarbons.

Sixteen halogenated aliphatic hydrocarbons were assayed for genotoxicity using the Ara mutagenicity assay with Salmonella typhimurium. Seven substances (1,2-dibromo-3-chloropropane, 1,2-dibromoethane, 1,2-dichloroethane, vinyl bromide, hexachloro-1,3-butadiene, iodoform and vinilydene chloride) were mutagenic at non-lethal doses. Comparatively, nine compounds (chloroform, carbon tetrachloride, 1,1,1-trichloroethane, 1,1,2-trichloroethane, tetrachloroethylene, trichloroethylene, 1,1,1,2-tetrachloroethane, 1,1,2,2-tetrachloroethane and hexachloroethane) were non-mutagenic after being assayed both in the presence and absence of metabolic activation with a rat liver microsomal fraction (S9). All negative compounds (except hexachloroethane) gave a lethal response, which could be an indication that bacteria were adequately exposed. The concordance between mutagenicity in the Ara test and carcinogenicity in rodents for this group of halogenated hydrocarbons was (31%) significantly lower than the concordance (72%) previously found in the Ara test with respect to a wider range of chemical classes. This result is in agreement with data reported for other genotoxicity assays. The presence of non-genotoxic carcinogens versus genotoxic non-carcinogens is discussed as a possible explanation. Five positive compounds (1,2-dibromo-3-chloropropane, 1,2-dibromoethane, 1,2-dichloroethane, vinyl bromide and hexachloro-1,3-butadiene) were analyzed for a quantitative relationship between carcinogenic potency in rats and the potency of response in the Ara mutagenicity test. This was possible because the Ara test, for volatile compounds (such as vinyl bromide), did not require the use of special vaporization techniques, which are difficult to evaluate quantitatively for mutagenic activity. A highly significant correlation was found between the mutagenic efficiencies of the five compounds in the Ara test and their carcinogenic potencies in rats.(ABSTRACT TRUNCATED AT 250 WORDS)