Dominique Fortini

Two Sibling Species of the Botrytis cinerea Complex, transposa and vacuma, Are Found in Sympatry on Numerous Host Plants

ABSTRACT Strains of Botrytis cinerea (the anamorph of Botryotinia fuckeliana) were collected from 21 different plant species around vineyards in the Champagne region (France). Strains were analyzed using three new polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) markers that were found by SWAPP (sequencing with arbitrary primer pairs), in addition to 15 other markers (PCR-RFLP, transposable elements, and resistance to fungicides). The markers revealed a high degree of genetic diversity and were used to investigate population structure. The two sympatric species transposa and vacuma, previously identified on grapes in these vineyards, were also detected on many of the plant species sampled. A new type of strain was also detected, having only the transposable element Boty. We did not detect any differentiation between strains from different organs or locations, but the prevalences of transposa and vacuma were significantly different on the different host plants. Fungicide resistance frequencies were significantly different in transposa and vacuma species. This study confirms that B. cinerea is a complex of sibling species and shows that the sibling species occur sympatrically on many host plants. However, the two species seemed to have different pathogenic behaviors. These findings contradict the traditional view of B. cinerea as a clonal population without specialization.

FLIPPER, A MOBILE FOT1-LIKE TRANSPOSABLE ELEMENT IN BOTRYTIS CINEREA

Abstract A transposable element, Flipper, was isolated from the phytopathogenic fungus Botrytis cinerea. The element was identified as an insertion sequence within the coding region of the nitrate reductase gene. The Flipper sequence is 1842 bp long with perfect inverted terminal repeats (ITRs) of 48 bp and an open reading frame (ORF) of 533 amino acids, potentially encoding for a transposase; the element is flanked by the dinucleotide TA. The encoded protein is very similar to the putative transposases of three elements from other phytopathogenic fungi, Fot1 from Fusarium oxysporum, and Pot2 and MGR586 from Magnaporthe grisea. The number of Flipper elements in strains of B. cinerea varied from 0 to 20 copies per genome. Analysis of the descendants of one cross showed that the segregation ratio of Flipper elements was 2:2 and that the copies were not linked.

Characterization of Bc-hch, the Botrytis cinerea homolog of the Neurospora crassa het-c vegetative incompatibility locus, and its use as a population marker

The Botrytis cinerea homolog (Bc-hch) of Nc-het-c and Pa-hch (vegetative incompatibility loci of Neurospora crassa and Podospora anserina respectively) was cloned and sequenced. The gene structure of Bc-hch is very close to those of Nc-het-c and Pa-hch. A PCR-RFLP approach on a 1171 bp fragment was used to screen polymorphism at this locus among 117 natural isolates of B. cinerea. Restriction patterns by the restriction enzyme HhaI fell into two allelic types. Moreover, haplotypes at the Bc-hch strictly corresponded to the resistance phenotypes to fenhexamid, a novel Botryticide. The use of Bc-hch as a population marker thus reveals a new structuring of B. cinerea natural populations into two groups (I and II). This result was confirmed by genic differentiation tests performed with five other markers on a sample of 132 B. cinerea isolates from the French region of Champagne.

Transformation of Botrytis cinerea with the nitrate reductase gene (niaD) shows a high frequency of homologous recombination

Abstract The nitrate reductase (niaD) gene was isolated from the phytopathogenic ascomycete Botrytis cinerea using a probe obtained by a polymerase chain reaction (PCR) with degenerate oligonucleotides corresponding to domains conserved among three fungal nitrate reductases. The B. cinerea niaD gene encodes a predicted protein of 907 amino acids and contains no intron. Nitrate reductase-deficient mutants of B. cinerea have been isolated. One of them was transformed with the niaD genes of Fusarium oxysporum f.sp. melonis and B. cinerea. The transformation was always ectopic when the donor DNA originated from F. oxysporum, but there was 80% gene replacement when the donor DNA originated from B. cinerea.

Expressed sequence tags from the phytopathogenic fungus Botrytis cinerea

A large set of genes was identified in the phytopathogenic fungus Botrytis cinerea by using an expressed sequence tag approach. The fungus was grown in axenic culture and a cDNA library was produced. From this library, 6559 ESTs were obtained. The combined sequences represent 3026 unisequences that corresponds to approximately one-quarter of the estimated total number of genes in B. cinerea. Approximately 18% of the ESTs showed significant similarities with genes coding for proteins with known functions,~56% were similar to genes coding for proteins with unknown functions and ~26% were orphans. A substantial B. cinerea gene inventory including putative virulence factors was therefore obtained and is now available at the Génoplante-Info Database interface (http://urgi.infobiogen.fr///Projects/GPiDB/Interface/).

Larval Exposure to the Juvenile Hormone Analog Pyriproxyfen Disrupts Acceptance of and Social Behavior Performance in Adult Honeybees.

Background Juvenile hormone (JH) plays an important role in honeybee development and the regulation of age-related division of labor. However, honeybees can be exposed to insect growth regulators (IGRs), such as JH analogs developed for insect pest and vector control. Although their side effects as endocrine disruptors on honeybee larval or adult stages have been studied, little is known about the subsequent effects on adults of a sublethal larval exposure. We therefore studied the impact of the JH analog pyriproxyfen on larvae and resulting adults within a colony under semi-field conditions by combining recent laboratory larval tests with chemical analysis and behavioral observations. Oral and chronic larval exposure at cumulative doses of 23 or 57 ng per larva were tested. Results Pyriproxyfen-treated bees emerged earlier than control bees and the highest dose led to a significant rate of malformed adults (atrophied wings). Young pyriproxyfen-treated bees were more frequently rejected by nestmates from the colony, inducing a shorter life span. This could be linked to differences in cuticular hydrocarbon (CHC) profiles between control and pyriproxyfen-treated bees. Finally, pyriproxyfen-treated bees exhibited fewer social behaviors (ventilation, brood care, contacts with nestmates or food stocks) than control bees. Conclusion Larval exposure to sublethal doses of pyriproxyfen affected several life history traits of the honeybees. Our results especially showed changes in social integration (acceptance by nestmates and social behaviors performance) that could potentially affect population growth and balance of the colony.

Larval exposure to thiamethoxam and American foulbrood: effects on mortality and cognition in the honey bee Apis mellifera

Here, we examined the in vitro effects of co-exposure to a pathogen and a common neonicotinoid on honey bee larvae survival and on adult learning behavior following a standard olfactory conditioning procedure based on the proboscis extension response paradigm. We exposed or co-exposed honey bee larvae to American foulbrood and to sub-lethal doses of thiamethoxam (chronic exposure). Our results revealed no additive effects between the two stressors on larval mortality. However, the present work provides the first evidence of impaired learning and memory in adult bees that were fed thiamethoxam (0.6 ng/bee) during the larval stage. We also show no alterations in learning and memory in bees after infection with American foulbrood at the larval stage. The present study contributes to our knowledge of the sub-lethal effects of neonicotinoids on honey bee larvae and adults.

Early steps of cryopreservation of day one honeybee ( Apis mellifera ) embryos treated with low-frequency sonophoresis

Honeybees, major providers of pollination, are endangered in many areas. Embryo cryopreservation may be a very useful tool to maintain their genetic diversity. However, it is complex in insects, because embryos are chill sensitive and are surrounded by two protectant membranes, the chorion and vitelline. These membranes prevent penetration of cryoprotectant in the embryos. This study aimed to test different conditions of embryo preparation before cryopreservation, including low-frequency sonophoresis, a physical method of permeabilization, and passages through cryoprotectant solutions. Apis mellifera ligustica embryos were collected in artificial cell plugs 7.5 h after queens had been caged, in two different seasons (winter, spring) and were then incubated in vitro overnight (16.5 h). Embryos were individually sonicated and then incubated in three cryoprotectant baths (B1 = 10%, B2 = 20% and B3 = 40% of cryoprotectant) and quenched in liquid nitrogen. Artificial cell plugs and in vitro incubation device were efficient in producing future embryos hatching. Embryos stained ruby red with rhodamine B after sonophoresis treatment indicated that low-frequency ultrasound had permeabilized embryos. According to the treatment, different significant hatching rates were obtained after sonophoresis (up to 25%). After three cryoprotectant incubations, best hatching rates were obtained after 10 min in B1 and B2, and 40 s in B3. These results show that sonophoresis is an efficient tool to permeabilize the chorion and vitelline membrane of the day one honeybee embryo allowing a hatching rate of more than 20%. They also show that the season is an important variability factor.