Biology and biocontrol potential of Ampelomyces mycoparasites, natural antagonists of powdery mildew fungi

Historically, pycnidial fungi belonging to the genus Ampelomyces were among the first mycoparasites to be studied in detail and were also the first fungi used as biocontrol agents of plant parasitic fungi. The interactions between host plants, powdery mildew fungi and Ampelomyces mycoparasites are one of the most evident cases of tritrophic relationships in nature although their study has received little attention in fungal and plant ecology so far. Ampelomyces mycoparasites have now become one of the most advanced in terms of commercial development of a fungal biocontrol agent, although there is still a need for more development work to produce a product with reliability approaching that of conventional chemical treatments. This review summarizes the taxonomy, genetic diversity, life cycle, mode of action, natural occurrence, host range, biocontrol potential, mass production and commercialization of these mycoparasites and compares the biocontrol ability of Ampelomyces with that of other fungal antagonists of powdery mildews.     

Hyperparasites of the genus shape Ampelomyces on powdery mildew fungi in Serbia

From 1982 to 1996, in Serbia, 229 species of plants infected with powdery mildew were collected. Seventy-five species had hyperparasites of the genus Ampelomyces. Distribution analysis showed that hyperparasitism was greatest on plants from the families Asteraceae, Apiaceae and Fabaceae. Ampelomyces was not found on the family Poaceae. It was reported for the first time on the families Amygdalaceae, Cornaceae, Grossulariaceae, Plantaginaceae, Rhamnaceae and other 37 plant species. Hyperparasites of the genus Ampelomyces were found on 33 different species of fungi that are causal agents of powdery mildews. They are reported for the first time on nine species. Pycnidial size varied in the range 45–106 × 25.5–40.5 μm and conidia from 4.5–10.5 × 2.5–4.8 μm. This revised version was published online in June 2006 with corrections to the Cover Date.     

Microsatellite DNA markers for Plasmopara viticola,the causal agent of downy mildew of grapes

Microsatellite loci were isolated from Plasmopara viticola (Oomycetes), the causal agent of downy mildew of grape, one of the most damaging fungal diseases of grapevine worldwide. Seven polymorphic loci were obtained from an enriched partial genomic library. A low genetic diversity was observed at all loci, with a mean observed allele number of 3.75 and an observed heterozygosity ranging from 0.074 to 0.547. Cross‐amplification tests on three closely related taxa indicated that two loci could be used in other Oomycetes species. These microsatellite loci were proved to be useful for population genetic analysis.     

Isolation of polymorphic microsatellite loci in the ponerine ant Ectatomma tuberculatum

We report the development of 11 microsatellite markers by an enrichment protocol in the ponerine ant Ectatomma tuberculatum. Polymorphism was explored in two colonies collected from Mexico and two colonies from Brazil. From the 11 loci which amplified, seven loci showed intracolonial polymorphism in Mexican colonies and only six loci were variable in populations from Brazil. Observed heterozygosity ranged from 0.18 to 0.84. The other five loci exhibited different alleles between Mexico and Brazil but geographical variability was not investigated further. Cross‐amplification was tested in another species of the same Tribe (Gnamptogenys striatula) and one locus was revealed to be polymorphic.     

Natural occurrence of ampelomyces intracellular mycoparasites in mycelia of powdery mildew fungi

This is the first extensive investigation that quantifies natural mycoparasitic relationships in the phyllosphere. The presence of Ampelomyces spp. was quantified in naturally occurring powdery mildew fungi collected in Hungary and Romania between 1992 and 1995. A total of 570 samples was studied representing 27 species (nine genera) of the Erysiphaceae infecting 41 host plant genera. The incidence of Ampelomyces spp., determined as the proportion of samples in which intracellular pycnidia were present, varied between 4.3 and 68.8% in the host fungal genera studied. The intensity of mycoparasitism, defined as a percentage of the powdery mildew mycelia parasitized by Ampelomyces , ranged from 0.15 to 65%. Both the incidence and the intensity of mycoparasitism showed the lowest values in Blumeria graminis (DC.) Speer collected from wild and cultivated monocotyledons, while the highest values were found in Arthrocladiella mougeotii (Lév.) Vassilkov infecting Lycium halimifolium Mill. plants. The paper reports for the first time the natural occurrence of Ampelomyces in Sawadaea bicornis (Wallr.:Fr.) Homma on maple.     

Characterization of polymorphic microsatellite DNA markers in Hakea oldfieldii Benth. (Proteaceae)

A genomic library was constructed and 13 polymorphic microsatellite markers were developed for Hakea oldfieldii, a woody shrub endemic to southwest Western Australia. Polymorphism was investigated for these markers in 28 individuals from a single population located in restricted habitat at the base of the Whicher Range south of Busselton. Expected heterozygosity ranged from 0.279 to 0.770 and averaged 0.633. Observed heterozygosity ranged from 0.321 to 0.786 and averaged 0.598. The number of alleles per locus ranged from 2.0 to 6.0 and averaged 4.5. These markers will be used to assay genetic diversity and pollen dispersal in this species.     

Characterization of porcine polymorphic microsatellite loci

Twenty-seven (CA)_n and two (GA)_n microsatellite clones were isolated out of a size-selected genomic pig library. These were sequenced and the number of uninterrupted dinucleotides was found to range from 12 to 26. Flanking primers were chosen for 11 dinucleotide repeats and optimal conditions for polymerase chain reaction (PCR) amplifications were established. Different microsatellite loci were amplified simultaneously by combining primer sets. Related and unrelated pigs were screened for length polymorphisms of the different microsatellite loci. The polymorphic information content (PIC) of these loci ranged between 0.62 and 0.83. Segregation studies in pig reference families established Mendelian inheritance. Locus S0022 was found to be X-linked.     

Characterization of microsatellite loci isolated from the blacktip shark and their utility in requiem and hammerhead sharks

We isolated and characterized 16 microsatellite loci from the blacktip shark, Carcharhinus limbatus, and tested cross‐species amplification in 11 Carcharhinus species and five additional shark genera. Thirty‐six (1.6%) and 180 (48%) colonies were positive for dinucleotide repeat motifs from unenriched and enriched libraries, respectively. Heterozygosities of polymorphic loci ranged from 0.04 to 0.96 with two to 22 alleles per locus. Amplification products were observed at nine to 13 loci (five to 11 of which where polymorphic) in 10 Carcharhinus species. Several loci were also polymorphic in each of the additional genera examined.     

PCR primers for polymorphic microsatellite loci in the desert locust,Schistocerca gregaria (Orthoptera: Acrididae)

Nine pairs of polymerase chain reaction (PCR) primers that amplify polymorphic microsatellite loci in the desert locust, Schistocerca gregaria (Forskal), were developed using a magnetic bead‐based enrichment protocol. A sample of 48 locusts collected during the 1993 and 1995 upsurge periods in Eritrea, East Africa, were genotyped. The number of alleles per locus ranged from six to 20; the average was 12.67. Allelic distributions were significantly different between samples from different localities.     

Management of powdery mildew and gray mold of cucumber by Trichoderma harzianum T39 and Ampelomyces quisqualis AQ10

Two biocontrol preparations were tested for their ability to control Sphaerotheca fusca and Botrytis cinerea on greenhouse cucumber. Trichoderma harzianum T39 (TRICHODEX) spray reduced powdery mildew severity by up to 97% but its efficacy declined to 18–55% control as the epidemic progressed. Unlike on young leaves, on older leaves the control of powdery mildew by T. harzianum T39 was poor. Ampelomyces quisqualis (AQ10) was very effective against powdery mildew, achieving up to 98% of control. Its effectiveness declined with the progress of the epidemic but unlike the other biocontrol agent it retained significant control capability on older leaves. Two aliphatic petroleum distillate oil products improved the efficacy of both biocontrol agents. The co-application of T. harzianum T39 and A. quisqualis AQ10 was tested on cucumber plants infected with powdery mildew followed by fruit gray mold infection. It resulted in no improvement of the control of powdery mildew, and in an improvement of gray mold control, the latter probably because of the use of additive oil (ADDQ) along with the second biocontrol preparation. There was no significant interference between the biocontrol agents in the co-application treatment as compared with the application of each agent alone; the level of population of T. harzianum T39 remained similar and the parasitism of S. fusca by A. quisqualis was not nullified. The application of T. harzianum T39 to soil instead of spraying it resulted in 75–90% lower powdery mildew coverage on the leaves. It was concluded that the mode of action of T. harzianum T39 in powdery mildew control is induced resistance, not mycoparasitism or antibiotic action.     

Highly polymorphic microsatellite loci in the facultatively eusocial hover wasp,Liostenogaster flavolineata and cross‐species amplification

We describe eight polymorphic microsatellite loci for facultatively eusocial hover wasps (Hymenoptera: Stenogastrinae). Loci were isolated from a partial genomic library of Liostenogaster flavolineata DNA, enriched for GA repeats and an unenriched library of Liostenogaster vechti DNA. We present our results of cross‐species amplification in three species: L. flavolineata, L. vechti and Parischnogaster alternata.     

A set of new microsatellite loci isolated from Chinese mitten crab,Eriocheir sinensis

Eriocheir sinensis has higher commercial value as food source than any other species of Eriocheir in China. To protect its germplasm resources and evaluate impact on local ecology, molecular tools to characterize the population structure and identify kinships from different water systems are highly needed. We have newly identified a set of 18 microsatellite markers with the observed number of alleles at each locus ranges from 8 to 30, the observed heterozygosity ranges from 0.140 to 0.900. These newly isolated markers increase the available molecular resources that can be used to analyse population structure of Chinese mitten crab.     

Characterization of microsatellite loci in White‐chinned Petrel (Procellaria aequinoctialis) and cross‐amplification in six other procellariiform species

Six polymorphic dinucleotide microsatellite loci were isolated and characterized from the White‐chinned Petrel Procellaria aequinoctialis, using a degenerate primer and PCR‐based technique to construct and screen an enriched genomic library. Preliminary data on three populations show heterozygosity levels ranging from 0.22 to 0.67 and allele numbers from three to nine. Preliminary data also suggest genetic distance between these three populations (F_ST 0.088). Cross‐species amplification of these six microsatellite loci and one further locus were tested in six other procellariiform species of the genus Procellaria, Macronectes, Thalassarche and Diomedea.     

Characterization of polymorphic microsatellite loci in Yucca filamentosa

Yuccas and their pollinator moths are a textbook example of mutualism, yet we lack sufficiently variable markers to properly study the population genetics of the plants. We characterized 13 polymorphic microsatellite loci for Yucca filamentosa by screening primers derived from an expressed sequence tag database. We found four to 13 alleles per locus and the observed heterozygosity ranged from 0.31 to 1. These markers will be useful in future ecological studies of Y. filamentosa.     

Characterization of nine polymorphic microsatellite loci from the alpine accentor Prunella collaris

Nine polymorphic microsatellite loci were isolated and characterized from the alpine accentor Prunella collaris. In analyses of 50 unrelated individuals, allelic diversity ranged from 2 to 7, and observed heterozygosity ranged from 0.08 to 0.70. Except for one locus (Pco2), variability was relatively low even in long repeat loci. This result may be due to the high return rate to the natal population. Primer sets were also tested in Japanese accentor Prunella rubida, and seven out of nine loci were successfully amplified.     

PERMANENT GENETIC RESOURCES: Isolation and characterization of polymorphic trinucleotide and dinucleotide microsatellite loci for the ice goby, Leucopsarison petersii

Eleven microsatellite markers were isolated and characterized for the ice goby, Leucopsarion petersii, from genomic libraries enriched for (ATG)(9) and (CA)(16) . Twenty individuals from a single population were used to screen polymorphism in these loci. The number of alleles per locus and observed heterozygosity ranged from four to 22 and from 0.35 to 1.00, respectively. All loci did not significantly deviated from Hardy-Weinberg equilibrium, and there was no evidence of linkage disequilibrium between all loci-pairs. These loci showed Mendelian inheritance in a full-sib family. The high level of polymorphism of these loci will be useful for studies of population genetics.     

Characterization of nine novel microsatellite markers from Brandt's vole (Lasiopodomys brandtii)

Highly polymorphic microsatellite markers are the most powerful tools to infer kinship relations. In this study, a library enriched for (AC)(n) (AG)(n) and (AGAT)(n) was constructed for screening microsatellite markers in Brandt's vole (Lasiopodomys brandtii), and nine novel polymorphic microsatellite markers were developed and characterized. The number of alleles ranged from 4 to 11 per locus and the mean polymorphism information content was 0.7535. The observed and the expected heterozygosity values averaged 0.760 (0.554-0.908) and 0.7914 (0.718-0.845), respectively. Average nonexclusion probability for one candidate parent varied from 0.485 to 0.716. These nine novel markers are highly polymorphic and powerful enough for our future kinship analysis.     

Characterization of polymorphic microsatellite DNA markers in Banksia nivea, formerly Dryandra nivea

A genomic library was constructed and 10 novel polymorphic microsatellite markers were developed for Banksia nivea subsp. uliginosa, a woody shrub endemic to the southwest of Western Australia. Polymorphism was investigated for these markers in 30 individuals from one population located in restricted habitat at the base of the Whicher Range east of Busselton. Expected heterozygosity ranged from 0.124 to 0.898 and averaged 0.570. Observed heterozygosity ranged from 0.133 to 0.933 and averaged 0.544. The number of alleles per locus ranged from 2.0 to 16.0 and averaged 7.3. These markers also produced amplification products in three other Banksia species and will be used to assay genetic diversity in these species.     

A novel strain of Pseudozyma aphidis from mulberry parasitises the conidia of mulberry powdery mildew fungus Phyllactinia sp. and its biocontrol effect in the fields

Plant surface is colonised with a vast community of non-pathogenic epiphytic microorganisms which play an important role in host defence. In the present study, we reported a fungus from mulberry leaf surface that showed an antagonistic effect against mulberry powdery mildew fungal pathogen Phyllactinia sp. This novel isolate is a yeast-like fungus that was identified as Pseudozyma aphidis CNm2012 based on morphologic and phylogenetic analysis. According to our research, P. aphidis CNm2012 directly acted on the powdery mildew conidia via parasitism which caused conidial atrophy, collapse and eventually, cleavage and death. During the parasitic process, we found the isolate could gather around the conidia of Phyllactinia sp. and form hyphae that grew on the conidial surface and utilise the conidia as a nutrient source. Field studies revealed that P. aphidis CNm2012 could suppress the disease incidence of mulberry powdery mildew caused by Phyllactinia sp., and reduce the disease severity. To our knowledge, it is the first report of P. aphidis directly against powdery mildew fungus Phyllactinia spp. by parasitism. Our results indicated that P. aphidis CNm2012 could be served as an environmentally friendly alternative of chemical pesticides to manage mulberry powdery mildew disease.     

Characterization of 16 polymorphic microsatellite loci in weathervane scallop Patinopecten caurinus

Discrete and commercially important patches of weathervane scallops (Patinopecten caurinus) are found in the northeastern Pacific Ocean. Little is known about weathervane scallop life history and population structure, but the species is vulnerable to overexploitation because of their large size and long lifespan. Here we describe 16 polymorphic microsatellite loci developed to study the relatedness of these discrete patches. All loci were polymorphic in the 32 individuals tested; the number of alleles range from four to 26 and heterozygosities ranged from 0.437 to 1.000.