Characterisation of Australian isolates of Fusarium oxysporum f. sp cubense by DNA fingerprinting analysis

Genetic variation among Australian isolates of the fungus Fusarium oxysporum f. sp. cubense (Foc), which causes Fusarium wilt in banana, was examined using DNA amplification fingerprinting (DAF). Ninety-four isolates which represented Races 1, 2, 3, and 4, and vegetative compatibility groups (VCGs) 0120, 0124, 0125, 0128, 0129, 01211, 01213/16, and 01220 were analysed. The genetic relatedness among isolates within each VCG, and between the 8 different VCGs of Foc present in Australia was determined. The DNA fingerprint patterns were VCG-specific, with each VCG representing a unique genotype. The genetic similarity among isolates within each VCG ranged from 97% to 100%. Among the different VCGs of Foc, 3 major clusters were distinguished which corresponded with race. All Race 1 and 2 isolates (VCGs 0124, 0125, 0128, and 01220) were closely related and clustered together, the Race 3 isolates from Heliconia clustered separately, and all Race 4 isolates (VCGs 0120, 0129, 01211, and 01213/16) clustered together. Fifteen isolates from Alstonville, NSW, were characterised because although they were classified as Race 2 based on their recovery from cooking banana cultivars, they belonged in VCG 0124, which had previously contained only Race 1 isolates. The occurrence of more than one race within a VCG means that vegetative compatibility grouping cannot be used to assign pathotype to pathogenic race as previously thought. It was possible to distinguish the Race 1 and Race 2 isolates within VCG 0124 using DNA fingerprinting, as each race produced a unique DNA fingerprint pattern. Among the Australian isolates, DNA fingerprinting analysis identified 9 different VCGs and genotypes of Foc.

Transfer of an autonomously replicating vector between vegetatively incompatible biotypes of Colletotrichum gloeosporioides

Previous research has indicated that biotypes A and B of Colletotrichum gloeosporioides that infect Stylosanthes spp. in Australia are asexual and vegetatively incompatible. Selectable marker genes conferring resistance either to hygromycin or phleomycin were introduced into isolates of these biotypes. Vectors conferring resistance to hygromycin and carrying telomeric sequences from Fusarium oxysporum replicated autonomously in C. gloeosporioides and gave frequencies of transformation 100-times higher than vectors that integrated into the genome. Monoconidial colonies resistant to both antibiotics were recovered when hygromycin-resistant biotype-A transformants carrying an autonomously replicating vector were paired in culture with a phleomycin-resistant biotype-B transformant carrying integrative vector sequences. Molecular analysis of double antibiotic-resistant progeny indicated that they contained the autonomous vector in a biotype-B genetic background, Results indicate that transfer of the autonomous vector had occurred from biotype A to biotype B, demonstrating the potential for transfer of genetic information between these biotypes.

Molecular characterization by amplified fragment length polymorphism and aflatoxin production of Aspergillus flavus isolated from freshly harvested peanut in Brazil

Brazil contributes substantially to the global peanut production, and the state of Sao Paulo is the largest producer in the country. Peanut crops can be contaminated by Aspergillus flavus strains producing aflatoxins, which are highly toxic and carcinogenic. Thus, the production of high-quality peanuts is crucial both for the commercial peanut industry and as a matter of public health. In this study, we used amplified fragment length polymorphism analysis (AFLP) to investigate the genetic variability among A. flavus strains isolated from fresh peanuts harvested in four different regions in the state of Sao Paulo, and to determine whether the molecular genetic profiles correlated with aflatoxin production or sclerotia formation. AFLP analysis generated 78 fragments ranging from 27 to 365 base pairs in length. Thirteen percent were not polymorphic. Genotyping identified twelve groups of A. flavus. On the basis of the polymorphisms identified, similarity between the isolates ranged from 37% to 100%. Of all isolates collected, 91.7% produced aflatoxins and 83.9% produced small sclerotia. Statistical analysis failed to suggest any relationship between the presence of sclerotia and mean levels of aflatoxins B-1 and B-2. Furthermore, a dendrogram based on AFLP data revealed substantial genetic variability among the A. flavus strains, but showed no correlation between dendrogram groups separated by molecular genetic features and production of aflatoxins B-1 or B-2 or the formation of sclerotia.

Identification of Aspergillus flavus isolates as potential biocontrol agents of aflatoxin contamination in crops

Aspergillus flavus, a haploid organism found worldwide in a variety of crops, including maize, cottonseed, almond, pistachio, and peanut, causes substantial and recurrent worldwide economic liabilities. This filamentous fungus produces aflatoxins (AFLs) B1 and B2, which are among the most carcinogenic compounds from nature, acutely hepatotoxic and immunosuppressive. Recent efforts to reduce AFL contamination in crops have focused on the use of nonaflatoxigenic A. flavus strains as biological control agents. Such agents are applied to soil to competitively exclude native AFL strains from crops and thereby reduce AFL contamination. Because the possibility of genetic recombination in A. flavus could influence the stability of biocontrol strains with the production of novel AFL phenotypes, this article assesses the diversity of vegetative compatibility reactions in isolates of A. flavus to identify heterokaryon self-incompatible (HSI) strains among nonaflatoxigenic isolates, which would be used as biological controls of AFL contamination in crops. Nitrate nonutilizing (nit) mutants were recovered from 25 A. flavus isolates, and based on vegetative complementation between nit mutants and on the microscopic examination of the number of hyphal fusions, five nonaflatoxigenic (6, 7, 9 to 11) and two nontoxigenic (8 and 12) isolates of A. flavus were phenotypically characterized as HSI. Because the number of hyphal fusions is reduced in HSI strains, impairing both heterokaryon formation and the genetic exchanges with aflatoxigenic strains, the HSI isolates characterized here, especially isolates 8 and 12, are potential agents for reducing AFL contamination in crops

Mapping the I-3 gene for resistance to Fusarium wilt in tomato: application of an I-3 marker in tomato improvement and progress towards the cloning of I-3

Fusarium wilt of tomato, caused by the fungal pathogen, Fusarium oxysporum f. sp. lycopersici (Fol), is an economically damaging disease that results in huge losses in Australia and other countries worldwide. The I-3 gene, which confers resistance to Fol race 3, has been described in wild tomato, Lycopersicon pennellii, accessions LA716 and PI414773. We are pursuing the isolation of I-3 from LA716 by map-based cloning. We have constructed a high-resolution map of the I-3 region and have identified markers closely flanking I-3 as well as markers co-segregating with I-3. In addition, construction of a physical map based on these markers has been initiated. This review describes the context of our research and our progress towards isolating the I-3 gene. It also describes some important practical outcomes of our work, including the development and use of a PCR-based marker for marker-assisted selection for I-3, and the finding that the I-3 gene from LA716 is different to that from PI1414773, which we have now designated I-7. Tomato varieties combining I-3 and I-7 have been developed and are currently being introduced into commercial production to further safeguard tomato crops against Fusarium wilt.

Mitochondrial inheritance during a parasexual cycle in {\it Fusarium oxysporum\/} forma specialis {\it cubense\/}

Fusarium oxysporum is a diverse, asexual fungal species composed of both saprophytic and pathogenic members. The destructive phytopathogens are classified into formae speciales based on the host species and into vegetative compatibility groups (VCGs) based on the ability of two individuals to form heterokaryons. Parasexuality, a non-sexual mode of genetic exchange unique to some fungi has been demonstrated in the laboratory in Fusarium oxysporum f. sp. cubense (FOC). The goals of this dissertation were threefold: to ascertain whether mitochondrial (mt) markers can distinguish race differences in FOC; to determine genetic relatedness of VCGs in FOC based on a mt marker; and to discover the mode of mt inheritance during a parasexual cycle.^ Band patterns produced by electrophoresis of Hae III digested genomic DNA indicated that VCG differences, not race, could be discerned by mtDNA analysis. Primers were designed to amplify a mt intergenic locus which served as a molecular marker to screen 55 strains of FOC in 16 VCGs using both single strand conformational polymorphism and DNA sequencing. Based on homogeneity of the locus, strains were assigned to seven mitotypes, a classification unit which I introduced and found informative for grouping related VCGs.^ To determine the mode of mt inheritance during a parasexual cycle, strains in different mitotypes were paired. Mitochondrial inheritance in all hybrid progeny was found to be uniparental. I speculated that if a parasexual cycle occurs in nature there would be greater variation in the nuclear genome than the mt. This could produce multiple VCGs within a mitotype, a phenomenon observed in FOC. Based on these data, I concluded that parasexuality in nature may contribute to the diversity observed in Fusarium oxysporum. ^

Horizontal genetic transfer in asexual fungi

Four aspects of horizontal genetic transfer during heterokaryon formation were examined in the asexual pathogen Fusarium oxysporum f.sp. cubense (Foc): (1) variability based on method of heterokaryon formation; (2) differences in nuclear and mitochondrial inheritance; (3) the occurrence of recombination without nuclear fusion; (4) the occurrence of horizontal genetic transfer between distantly related isolates. The use of non-pathogenic strains of Fusarium oxysporum as biocontrol agents warrants a closer examination at the reproductive life cycle of this fungus, particularly if drug resistance or pathogenicity genes can be transmitted horizontally. Experiments were divided into three phases. Phase I looked at heterokaryon formation by hyphal anastomosis and protoplast fusion. Phase II was a time course of heterokaryon formation to look at patterns of nuclear and mitochondrial inheritance. Phase III examined the genetic relatedness of the different vegetative compatibility groups using a multilocus analysis approach. Heterokaryon formation was evident within and between vegetative compatibility groups. Observation of non-parental genotypes after heterokaryon formation confirmed that, although a rare event, horizontal genetic transfer occurred during heterokaryon formation. Uniparental mitochondria inheritance was observed in heterokaryons formed either by hyphal anastomosis or protoplast fusion. Drug resistance was expressed during heterokaryon formation, even across greater genetic distances than those distances imposed by vegetative compatibility. Phylogenies inferred from different molecular markers were incongruent at a significant level, challenging the clonal origins of Foc. Mating type genes were identified in this asexual pathogen Polymorphisms were detected within a Vegetative Compatibility Group (VCG) suggesting non-clonal inheritance and/or sexual recombination in Foc. This research was funded in part by a NIH-NIGMS (National Institutes of Health-National Institute of General Medical Sciences) Grant through the MBRS (Minority Biomedical Research Support), the Department of Biological Sciences and the Tropical Biology Program at FIU. ^

Analysis of Heterokaryon Formation in Fusarium oxysporum f. sp. cubense.

Fusarium oxysporum forma specialis cubense is a soilborne phytopathogen that infects banana. The true evolutionary identity of this so called species, Fusarium oxysporum, is still unknown. Many techniques have been applied in order to gain insight for the observed genetic diversity of this species. The current classification system is based on vegetative compatibility groups (VCG's). Vegetative compatibility is a self non-self recognition system in which only those belonging to a VCG can form stable heterokaryons, cells containing two distinct nuclei. Heterokaryons in turn, are formed from hypha! anastomosis, the fusion of two hyphae. Furthermore, subsequent to heterokaryon formation potential mechanisms exist which may generate genetic variability. One is through viral transfer upon hyphal anastomosis. The other mechanism is a form of mitotic recombination referred to as the parasexual cycle. Very little research has been performed to directly obser.ve the cellular events; hypha! anastomosis, heterokaryon formation, and the parasexual cycle in Fusarium oxysporum f. sp. cubense. The purpose of this research was to design and use methods which would allow for the detection of hypha! anastomosis and heterokaryon formation, as well as any characteristics surrounding this event, within and between VCG's in Foe. First, some general growth properties were recorded: the number of nuclei per hypha, the size ofthe hyphal tip cell, the size of the cell adjacent to the hypha! tip (pre-tip) cell, and the number of cells to the first branch point. Second, four methods were designed in order to assay hyphal anastomosis and heterokaryon formation: 1) pairings on membrane: phase or brightfield microscopy, 2) pairings on membrane: fluorescence microscopy, 3) spore crosses: fluorescence microscopy, and 4) double picks in fractionated MMA. All of these methods were promtsmg.

Agronomic performance of 27 cocoa progenies and plant selection based on productivity, self-compatibility and disease resistance

The objective of the present work was to evaluate 27 progenies of cocoa crosses considering the agronomic traits and select F1 plants within superior crosses. The experiment was installed in March 2005, in the Experimental Station Joaquim Bahiana (ESJOB), in Itajuipe, Bahia. The area of the experiment is of approximately 3 ha, with a total of 3240 plants. Thirteen evaluations of vegetative brooms, five of cushion brooms and 15 of number of pods per plant were accomplished. Thirty pollinations were made for each selected plant to test for self-compatibility. The production, based on the number of pods per plant, and resistance to witches´ broom indicated CEPEC 94 x CCN 10, RB 39 x CCN 51 and CCN 10 x VB 1151 as superior progenies. All selections tested were self-compatible. The analyses of progenies and individual tree data, associated to visual field observations, allowed the selection of 17 plants which were included in a network of regional tests to determine the phenotypic stability.

Semi-determinate Growth Habit Adjusts The Vegetative-to-reproductive Balance And Increases Productivity And Water-use Efficiency In Tomato (solanum Lycopersicum).

Tomato (Solanum lycopersicum) shows three growth habits: determinate, indeterminate and semi-determinate. These are controlled mainly by allelic variation in the SELF-PRUNING (SP) gene family, which also includes the florigen gene SINGLE FLOWER TRUSS (SFT). Determinate cultivars have synchronized flower and fruit production, which allows mechanical harvesting in the tomato processing industry, whereas indeterminate ones have more vegetative growth with continuous flower and fruit formation, being thus preferred for fresh market tomato production. The semi-determinate growth habit is poorly understood, although there are indications that it combines advantages of determinate and indeterminate growth. Here, we used near-isogenic lines (NILs) in the cultivar Micro-Tom (MT) with different growth habit to characterize semi-determinate growth and to determine its impact on developmental and productivity traits. We show that semi-determinate genotypes are equivalent to determinate ones with extended vegetative growth, which in turn impacts shoot height, number of leaves and either stem diameter or internode length. Semi-determinate plants also tend to increase the highly relevant agronomic parameter Brix×ripe yield (BRY). Water-use efficiency (WUE), evaluated either directly as dry mass produced per amount of water transpired or indirectly through C isotope discrimination, was higher in semi-determinate genotypes. We also provide evidence that the increases in BRY in semi-determinate genotypes are a consequence of an improved balance between vegetative and reproductive growth, a mechanism analogous to the conversion of the overly vegetative tall cereal varieties into well-balanced semi-dwarf ones used in the Green Revolution.

Tissue culture of Cecropia glaziovii Sneth (urticaceae): vegetative micropropagation and plant regeneration from callus

Cecropia glaziovii is a tree with used in Brazilian popular medicine. Methods allowing the clonal propagation of this species are of great interest for superior genotype multiplication and perpetuation. For this reason, we examined the effect of different culture media and different types of explants on adventitious shoot regeneration from callus and buds of C. glaziovii. Leaves, petioles and stipules obtained from aseptically grown seedlings or from pre-sterilized plants were used to initiate cultures. Adventitious shoot regeneration was achieved when apical and axillary buds were inoculated on gelled Murashige & Skoog (MS) medium supplemented with 6-benzylaminopurine alone (BAP) (1.0, 5.0 or 10.0 mg L-1) or combined with -naphthalene acetic acid (NAA) (1.0 or 2.0 mg L-1), after 40 days of culture. Best callus production was obtained after 30 days of petioles' culture on gelled MS medium with 2,4 dichlorophenoxyacetic acid (2,4-D) (5.0 mg L-1) combined with BAP (1.0 mg L-1). Successful shoot regeneration from callus was achieved when MS medium supplemented with zeatin (ZEA) (0.1 mg L-1) alone or combined with 2,4-D (1.0 or 5.0 mg L-1) was inoculated with friable callus obtained from petioles. All shoots were rooted by inoculation on MS medium supplemented with indole-3-acetic acid (IAA) (1.0 mg L-1). Rooted plants transferred to potting soil were successfully established. All in vitro regenerated plantlets showed to be normal, without morphological variations, being also identical to the source plant. Our study has shown that C. glaziovii can be propagated by tissue culture methods, allowing large scale multiplication of superior plants for pharmacological purposes.

Chemical changes in bromeliad leaves at different vegetative stages

Studies concerning the accumulating capacity of native epiphytic bromeliads are of utmost relevance, due to the continuous incorporation of chemical elements provided by these organisms in the ecosystems. Bromeliad species from diverse So Paulo State conservation units, Brazil, were sampled for young, mature and old leaves using a sustainable sampling method. By applying INAA, the accumulation of ten chemical elements, i.e. Br, Ca, Co, Fe, K, Na, Rb, Sc, Sr and Zn, was investigated in different leaf vegetative stages. The bromeliads showed divergent chemical element distribution patterns, demonstrating a real complexity in the accumulation and translocation mechanisms utilized by these plants.

Compatibility of Neoseiulus paspalivorus and Proctolaelaps bickleyi, candidate biocontrol agents of the coconut mite Aceria guerreronis: spatial niche use and intraguild predation

The eriophyid mite Aceria guerreronis occurs in most coconut growing regions of the world and causes enormous damage to coconut fruits. The concealed environment of the fruit perianth under which the mite resides renders its control extremely difficult. Recent studies suggest that biological control could mitigate the problems caused by this pest. Neoseiulus paspalivorus and Proctolaelaps bickleyi are two of the most frequently found predatory mites associated with A. guerreronis on coconut fruits. Regarding biological control, the former has an advantage in invading the tight areas under the coconut fruit perianth while the latter is more voracious on the pest mites and has a higher reproductive capacity. Based on the idea of the combined use/release of both predators on coconut fruits, we studied their compatibility in spatial niche use and intraguild predation (IGP). Spatial niche use on coconut fruits was examined on artificial arenas mimicking the area under the coconut fruit perianth and the open fruit surface. Both N. paspalivorus and P. bickleyi preferentially resided and oviposited inside the tight artificial chamber. Oviposition rate of P. bickleyi and residence time of N. paspalivorus inside the chamber were reduced in the presence of a conspecific female. Residence of N. paspalivorus inside the chamber was also influenced by the presence of P. bickleyi. Both N. paspalivorus and P. bickleyi preyed upon each other with relatively moderate IGP rates of adult females on larvae but neither species yielded nutritional benefits from IGP in terms of adult survival and oviposition. We discuss the relevance of our findings for a hypothetic combined use of both predators in biological control of A. guerreronis.

Vegetative and fruit characteristics of papaya trees obtained by mass selection

The present work had as purpose to evaluate some characteristics of papaya trees (Carica papaya L.), Golden cultivar, obtained trough plant mass selection, regarding plant and fruit quality in the first months of production. The samples were evaluated in a commercial crop at: 0, 20, 40, 70, 130, 180, 230, 260, 280, 310 and 340 days after the planting (DAP) and the first fruits were harvested at 230 DAP. The results showed the low height (199cm in 340 DAP) and low first flowering`s heigth (71cm), which is important to facilitate the harvest process. The plants presented good yield with high number of leafs (allowing a great area of fruit cover) and about 60 fruits per plant. The fruits kept similar features to cv. Golden. The fruit`s fresh weight ranged from 302.4 to 467.5g, which is in the range of the Brazilian market. The pulp thickness was 2.35cm, which is a feature of great economic interest. The pulp thickness showed close relation with climatic factors, and great variations of temperature and precipitation accelerated the pulp loss of thickness.