Species-specific Primer Pairs Research Articles

Development of a species‐specific AFLP‐based SCAR marker for authentication of black muntjac (Muntiacus crinifrons)

Black muntjac is a rare and endangered deer endemic to eastern China. Due to the economic and pharmaceutical value of the meat, antlers and skin, the species has chronically suffered from poaching though it is regarded as the state key protected animal. To provide an effective molecular method for authentication of tissue specimen (such as meat, skin etc.) of the species, we developed a Sequence Characterized Amplified Region (SCAR) derived from a species specific Amplification Fragment Length Polymorphism (AFLP) marker. Initially, a 707-bp species specific DNA fragment of the animal was detected by a pair of AFLP primers (Ep7/Mp8). Subsequently, a species-specific primer pair (P-F/P1-R) was designed based on the specific AFLP fragment sequence, obtaining a 298-bp SCAR for the species. Finally, the reliability of the SCAR primers was verified by two separate PCRs using the designed SCAR primers and a cyt b universal primer pair. As expected, all black muntjac samples presented two bands but the others failed to produce the SCAR by merely showing one band. Our results indicated that the SCAR primers developed in this study may provide a useful tool for forensic authentication of black muntjac samples though further testing with larger sample sizes is warranted.

Development of a multiplex loop-mediated isothermal amplification (mLAMP) method for the simultaneous detection of white spot syndrome virus and infectious hypodermal and hematopoietic necrosis virus in penaeid shrimp

A multiplex LAMP (mLAMP) assay was developed for the simultaneous detection of two penaeid shrimp viruses, namely, white spot syndrome virus (WSSV) and infectious hypodermal and hematopoietic necrosis virus (IHHNV), which are the major viral pathogens of penaeid shrimp worldwide. Two sets of the LAMP primers were designed for VP28 gene of WSSV and non-structural protein 1 (NS1) gene of IHHNV, in which a restriction enzyme cleavage site was inserted into two pairs of species-specific primers, to construct the mLAMP assay by combining these two sets totaling eight primers. The mLAMP method was distinguishable between WSSV and IHHNV due to the subsequent restriction enzyme analysis. The sensitivities of the mLAMP method were 102 and 103 times higher on the detection limits for WSSV and IHHNV, respectively, than those of the nested-PCR (nPCR) and classical PCR methods. No positive results were displayed when nucleic acids from other shrimp pathogen DNAs were used as mLAMP templates. Uracil-N-glycosylase (UNG) was used to prevent carry-over contamination of mLAMP products. In addition to its high specificity and sensitivity, the developed multiplex LAMP assay offers an efficient, convenient and rapid tool for screening penaeid shrimp viruses since both WSSV and IHHNV can be diagnosed in a single reaction, and will be useful for the control of these viruses in shrimp.

Detection of Leptosphaeria maculans from imported Canola seeds

Leptosphaeria maculans, the causal agent of blackleg on oilseeds in many regions around the world, has not been reported in China. In this study, L. maculans was detected from imported canola seeds by polymerase chain reaction (PCR) using a species-specific primer pair. The isolated L. maculans exhibited morphology identical to the known L. maculans strain on PDA culture plates and produced typical blackleg symptom when inoculated on oilseed leaves. It is the first report on the detection and identification of L. maculans from Canadian oilseed in China.

First Report of Anthracnose Caused by Colletotrichum acutatum on Persimmon Fruit in the United States.

Persimmon trees are important for their fruit as well as their colorful fruit and foliage in the fall. Persimmon fruit (Japanese persimmon, Diospyros kaki cv. Fuyu) were collected in November 2008 from a tree in Windsor, NC, located in the Coastal Plain. Fruit were not symptomatic on the tree but developed dark lesions after harvest. Isolations from six fruit yielded seven isolates of Colletotrichum acutatum J. H. Simmonds. After incubation at 25°C under continuous light for 15 days on potato dextrose agar (PDA), all isolates had gray aerial mycelium, but the inverse sides of the plates of six isolates were maroon and one was beige. Masses of salmon-colored conidia were formed first in the center of the colonies, then were observed scattered across the colonies in older cultures. Conidia were hyaline, one-celled, elliptic with one or both ends pointed, and measured 8.1 to 16.3 × 3.1 to 5 μm. Setae and sclerotia were not observed. There were also dark structures measuring 1 to 10 mm that were partially embedded in the agar that contained conidia. Cultural and conidial characteristics of the isolates were similar to those of C. acutatum (3). PCR amplification was performed with the species-specific primer pair CaInt2/ITS4 (2) and genomic DNA from the original isolates and isolates obtained from inoculated fruit. An amplification product of approximately 490 bp, which is specific for C. acutatum, was observed. To fulfill Koch's postulates, persimmon fruit obtained from the grocery store were surface disinfested with 0.5% sodium hypochlorite and sterile filter paper disks dipped in conidial suspensions (1 × 105 conidia/ml) of two C. acutatum isolates (maroon and beige reverse) or sterile, deionized water were placed on the fruit. Three fruit were inoculated per treatment and the disks were placed on four locations on each fruit. Parafilm was wrapped around the diameter of the fruit to keep the filter paper disks moist and in place. Fruit were placed in moist chambers and incubated at 25°C. After 3 days, the Parafilm was removed and the fruit returned to the moist chambers. Small, dark lesions were observed on fruit inoculated with each isolate of C. acutatum when the filter paper disks were removed. Ten days after inoculation, dark lesions and acervuli with salmon-colored masses of conidia were observed on fruit inoculated with both isolates of C. acutatum and the fruit were soft. After 12 days, there were abundant masses of conidia and the inoculated areas were decayed. Control fruit remained firm and did not develop symptoms. Cultures obtained from the fruit and the conidia produced were typical of the isolates used to inoculate the fruit. C. acutatum has been reported to cause fruit rot on persimmon fruit in New Zealand (1). To our knowledge, this is the first report of C. acutatum on persimmon fruit in the United States.

Mycotic DNA in Non-Atherosclerotic Aortic Wall of Coronary Patients Is Associated With sICAM-1 Expression

Atherosclerosis is currently being investigated as a chronic inflammatory process and the role of infectious agents is unclear. The presence of mycotic DNA in the wall of the non-atherosclerotic aorta of patients with coronary artery disease (CAD) and its association with levels of soluble intercellular adhesion molecule (sICAM)-1 expression was examined in the present study. In 40 patients with CAD and a comparative group of 20 patients with aortic valve stenosis (AS) without CAD, specimens of the aortic wall were obtained during cardiac surgery. Mycotic DNA was analyzed by polymerase chain reaction (PCR) using a fungus-specific universal primer pair, ITS3 and ITS4, to amplify a portion of the 5.8S rDNA region, the entire ITS2 region and a portion of the 28S rDNA region, and using a species-specific primer pair, CALB1 and CALB2, to specifically amplify Candida (C.) albicans. The nested PCR method was performed to amplify the intergenic transcribed spacer regions of the rRNAs of Candida species. Before surgery the serum level of sICAM-1 was estimated. Mycotic DNA was detected in 48% of the CAD patients and in 40% of the AS patients, with C. albicans DNA in 58% and 100%, respectively (P>0.05). In CAD patients with a high level of sICAM-1, C. albicans DNA was found more frequently than in patients without elevated levels of sICAM-1 (P<0.05). Mycotic DNA was found in the non-atherosclerotic aortic wall of CAD patients as well as in patients with AS. In the CAD patients C. albicans DNA was related to sICAM-1 expression.

Differentiation of sourdough yeast species by a novel species-specific PCR assay

The objective of this study was to develop species-specific primer pairs based on the internal transcribed spacer region (ITS) of the ribosome DNA for species identification of the frequently found sourdough yeast species. Species-specific primers were designed by the alignment of sourdough yeast ITS sequences, which were then employed for PCR using the template DNA of sourdough yeast strains. PCR primers were shown to be specific for the following species: Issatchenkia orientalis (Candida krusei), C. humilis, Kazachstaniaexigua (C. holmii), Pichia anomala and P. subpelliculosa. Therefore, we conclude that our novel species-specific primers could be used to rapidly and accurately identify the most frequent sourdough yeast species using a PCR-based assay.

Simultaneous Identification of 14 Genital Microorganisms in Urine by Use of a Multiplex PCR-Based Reverse Line Blot Assay

The aim of this study was to develop and evaluate a sensitive method for the simultaneous identification of 14 urogenital potential pathogens. A multiplex PCR-based reverse line blot (mPCR/RLB) assay was developed to detect 14 urogenital pathogens or putative pathogens, namely Trichomonas vaginalis, Streptococcus pneumoniae, Neisseria gonorrhoeae, Chlamydia trachomatis, Ureaplasma parvum, U. urealyticum, Gardnerella vaginalis, Haemophilus influenzae, herpes simplex virus type 1 (HSV1) and HSV2, N. meningitidis, Mycoplasma hominis, M. genitalium, and adenovirus, using two species-specific primer pairs and probes for each. The method was validated using a reference strain or a well-characterized clinical isolate of each target organism and was found to be both sensitive and specific. The limits of detection for the mPCR/RLB assay varied among the 14 target organisms from 4.2 x 10(-1) to 7.0 x 10(-11) ng/microl of genomic DNA. There were no cross-reactions among any of the probes. This method was used to test 529 first-voided urine specimens from male patients with and without urethritis attending two Sydney sexual health clinics. One or more target species were detected in 193 (36%) subjects. Of 233 positive results, overall 216 (93%) were concordant between mPCR/RLB and a comparator method (culture and/or species-specific PCR), 9 were positive only by mPCR/RLB, and 8 were positive only by the comparator method. The mPCR/RLB method was an accurate, convenient, and inexpensive method for the detection of multiple potential pathogens in first-voided urine specimens from men.

Differentiation of Two Pathogens of Powdery Mildew Disease in Flowering Dogwood (Cornus florida) by PCR-mediated Method Based on ITS Sequences

Two fungi, Phyllactinia guttata and Erysiphe pulchra were identified as the pathogens of powdery mildew of flowering dogwood (Cornus florida). The objective of this research was to identify and distinguish the two fungi by developing species-specific primers. The internal transcribed spacer (ITS) universal primers and a series of species-specific primers designed from the ITS regions were used to evaluate and validate the two fungi causing powdery mildew in dogwood. Four primer pairs showed specificity to P. guttata and three to E. pulchra. These species-specific primer pairs can be used as molecular markers to provide diagnostic tools for detection and differentiation of the two powdery mildew pathogens in flowering dogwood.

First Report of Bitter Rot Caused by Colletotrichum acutatum on Apple in China.

Bitter rot of apple caused by Colletotrichum gloeosporioides was first reported in China in 1985 (3). In China, apples are grown on approximately 2 million ha, and bitter rot occurs in almost all production areas, with crop damage ranging from 30 to 70%. During the summer of 2007, fungi were isolated from apple fruit exhibiting bitter rot symptoms in 12 and 9 orchards in Shaanxi and Henan provinces, respectively, in China. Symptoms included 2- to 3-cm-diameter, sunken, brown lesions on the fruit surface that contained black, pinhead-size fruiting structures producing orange conidial masses under high humidity, similar to that of C. gloeosporioides. On potato dextrose agar (PDA), colonies were white, pale gray, or pale orange when grown at 25°C. Conidia were 8 to 16 × 2.5 to 4 μm, fusiform, pointed at one or both ends, one celled, thin walled, aseptate, and hyaline. Appressoria were 6.5 to 11 × 4.5 to 7.5 μm, clavate to circular, and light to dark brown. These characteristics matched published descriptions of C. acutatum (2). To confirm pathogenicity, three mature, healthy apples (cv. Fuji) were surface disinfested with 70% ethanol and then wounded with a sterile needle. After being inoculated with a spore suspension (1 × 105 conidia/ml) prepared from a 2-week-old culture on PDA, these apples were sealed in a plastic bag and incubated at 25°C. Symptoms appeared 3 to 5 days after inoculation and began to enlarge 7 days later, forming lesions with fruiting structures. Under high humidity, cream-to-salmon pink spore masses were produced on lesions. As the lesions enlarged, the rot progressed to the core of the fruit in a V-shaped pattern. When the pathogen was reisolated from lesions of inoculated fruit onto PDA and incubated at 25°C, colony and conidial morphology were identical to those of the original isolates. Tests were performed three times with similar results. PCR with species-specific primer pair CaInt2/ITS4 (1) of genomic DNA from the isolates resulted in an amplification product of approximately 490 bp, which is specific for C. acutatum. The sequences exhibited 99% similarity with those of C. acutatum isolates AB273195 from GenBank. Approximately 20 of 103 symptomatic fruit from the field survey yielded fungal cultures whose morphology was consistent with that of C. acutatum, whereas the other cultures were C. gloeosporioides and Botryosphaeria dothidea. To our knowledge, this is the first report of bitter rot of apple caused by Colletotrichum acutatum in China.

Evaluation of different methods for the detection and identification of Enterobactersakazakii isolated from South African infant formula milks and the processing environment

Enterobacter sakazakii is an emerging pathogen associated with life-threatening neonatal infections resulting from the consumption of contaminated powdered infant formula milk (IFM). Recent taxonomic analyses have determined that E . sakazakii comprises a number of genomospecies, and it has been proposed that E . sakazakii be reclassified as a novel genus, “Cronobacter”. Accurate methods are required for the rapid detection and identification of this group of micro-organisms, since even low cell numbers have been reported to cause disease. The aim of this study was to evaluate various E . sakazakii detection methods in order to ascertain the most suitable method for detection and identification of these pathogenic agents. Samples from IFM and the environment were evaluated for the presence of E . sakazakii using the isolation steps (pre-enrichment, enrichment and selection) described in the Food and Drug Administration (FDA) method for E . sakazakii detection. Sixty-four isolates (50 from IFM and 14 from the environment) were selected from tryptone soy agar (TSA), regardless of colony appearance, and these isolates were identified by 16S ribosomal DNA (rDNA) sequencing. Thereafter, different culture-dependent and culture-independent methods were evaluated to accurately detect and identify the E . sakazakii isolates. These methods included the assessment of yellow pigment production on TSA, typical colonies on chromogenic Druggan–Forsythe–Iversen (DFI) and Chromocult® Enterobacter sakazakii (CES) media and polymerase chain reaction (PCR) using six different species-specific primer pairs described in the literature. Identification of E . sakazakii using yellow pigment production was demonstrated to have a low sensitivity, specificity and accuracy (87%, 71% and 74%, respectively), which lowers the suitability of the FDA method. Chromogenic DFI and CES media were sensitive, specific and accurate (100%, 98% and 98%, respectively) for the detection of E . sakazakii. The specificity of the PCR amplifications ranged from 8% to 92%, emphasising the need for rigorous primer testing against closely related species. Of the primer pairs evaluated, Esakf/Esakr were the most suitable for E . sakazakii detection and identification. The detection limit of Esakf/Esakr was found to be 10 4 CFU/ml. This study demonstrated that no single method was capable of unambiguously confirming the presence and identity of E . sakazakii isolates, that each method had inherent advantages and disadvantages, and that in most cases several methods were required for accurate detection and identification. Further, it was demonstrated that the current FDA method for E . sakazakii detection should be revised in the light of the availability of more sensitive, specific and accurate detection methods.

Identification of Heterodera glycines using PCR with sequence characterised amplified region (SCAR) primers

AbstractA single randomly amplified polymorphic DNA (RAPD) marker, OPA06477, species-specific to the soybean cyst nematode species, Heterodera glycines, was identified. After sequencing the RAPD-PCR products, primers of 24 nucleotides were designed to complement the terminal DNA sequence of the DNA fragments. This resulted in one pair of species-specific primers that were used to amplify the sequence characterised amplified regions (SCAR). The developed sets of SCAR primers were successfully used in straightforward, fast and reliable PCR assays to identify H. glycines. The SCAR markers can be amplified from DNA from single second-stage juveniles and females. This is the first time SCAR primers have been combined with universal primers D2A and D3B in order to avoid false negative results.

Differentiation of two powdery mildews of sunflower (Helianthus annuus) by a PCR-mediated method based on ITS sequences

Powdery mildew can be found in most sunflower fields during the winter season in Taiwan and causes severe yellowing on the blade, petiole, stem, and calyx, as well as serious defoliation. Two types of powdery mildew fungi isolated from sunflower leaves showed variable status for fibrosin bodies. But only the cleistothecium of Podosphaera xanthii, one of the pathogens causing this disease, was observed on samples from Chungpu County at the beginning of 2005. With a species-specific primer pair, PN23/PN34, no specific PCR product was amplified from the pathogen’s genomic DNA. Based upon the ITS sequence of rDNA, three PCR primer sets (S1/S2, G1/G2, and L1/L2) specific to P. xanthii, Golovinomyces cichoracearum and Leveillula taurica, respectively, were designed to detect and differentiate pathogens causing powdery mildews on sunflower. Only the primer pairs S1/S2 and G1/G2 could amplify PCR products, with product sizes of 454 and 391 bp, respectively. Four samples of fungal DNA were subjected to a multiplex PCR amplification with primer pairs S1/S2 and G1/G2; P. xanthii and G. cichoracearum were successfully detected. These results suggest that the multiplex PCR method is a rapid, simple, and effective technique to detect and differentiate powdery mildews, for example P. xanthii and G. cichoracearum, found on sunflower. With morphologic characteristics, ITS sequence analysis and pathogenicity testing, P. xanthii and G. cichoracearum, the first case, are two powdery mildews on sunflower in Taiwan.

Polymerase chain reaction (PCR) identification of Aspergillus niger and Aspergillus tubingensis based on the calmodulin gene

Aspergillus niger and A. tubingensis, species belonging to section Nigri, are commonly found in plant products and processed food, such as grapes, cereals, coffee, and derived products. These two species are very difficult to differentiate by classical morphological criteria and some isolates are known to produce ochratoxin A. The exact identification of these two species is very important to avoid the overestimation of toxicological contamination and related risks. A polymerase chain reaction (PCR)-based identification and detection assay was developed as a tool to identify A. niger and A. tubingensis, using molecular differences obtained by sequencing the calmodulin gene. Two pairs of species-specific primers were designed and empirically evaluated for PCR identification of A. niger and A. tubingensis. Species-specific PCR products generated by each primer set were 505 bp (A. tubingensis) and 245 bp (A. niger) in length, which could be potentially useful for a multiplex PCR assay. The sensitivity of this assay was about 10 pg DNA in a 25-µl PCR reaction volume, using pure total DNA of the two species. The method described in this study represents a rapid and reliable procedure to assess the presence in food products of two ochratoxigenic species of section Nigri.

Species identification of animal cells by nested PCR targeted to mitochondrial DNA

We developed a highly sensitive and convenient method of nested polymerase chain reaction (PCR) targeted to mitochondrial deoxyribonucleic acid (DNA) to identify animal species quickly in cultured cells. Fourteen vertebrate species, including human, cynomolgus monkey, African green monkey, mouse, rat, Syrian hamster, Chinese hamster, guinea pig, rabbit, dog, cat, cow, pig, and chicken, could be distinguished from each other by nested PCR. The first PCR amplifies mitochondrial DNA fragments with a universal primer pair complementary to the conserved regions of 14 species, and the second PCR amplifies the DNA fragments with species-specific primer pairs from the first products. The species-specific primer pairs were designed to easily distinguish 14 species from each other under standard agarose gel electrophoresis. We further developed the multiplex PCR using a mixture of seven species-specific primer pairs for two groups of animals. One was comprised of human, mouse, rat, cat, pig, cow, and rabbit, and the other was comprised of African green monkey, cynomolgus monkey, Syrian hamster, Chinese hamster, guinea pig, dog, and chicken. The sensitivity of the PCR assay was at least 100 pg DNA/reaction, which was sufficient for the detection of each species of DNA. Furthermore, the nested PCR method was able to identify the species in the interspecies mixture of DNA. Thus, the method developed in this study will provide a useful tool for the authentication of animal species.

MONITORING OF GLOBODERA SPP. IN LITHUANIA USING DIAGNOSTIC MORPHOMETRIC ANALYSIS AND POLYMERASE CHAIN REACTION

A total of 11,406 soil samples from 2,742 ha were collected in 10 administrative regions of Lithuania during the year 2006. A total of 672 cysts were selected and species were morphologically identified as Globodera rostochiensis. Of these cysts, 117 from 5 administrative regions of Lithuania were identified using polymerase chain reaction (PCR) analysis. Two pairs of species-specific primers were used to distinguish G. rostochiensis from G. pallida. This is the first application of a PCR method for identification of Globodera species in Lithuania.

Molecular identification of wolf spiders (Araneae: Lycosidae) by multiplex polymerase chain reaction

Ecological field studies are often hindered by the difficulty of identifying certain taxonomic groups. For example, no identification keys exist for immature Australian wolf spiders, which are important predators of insect pests. This makes it difficult to identify the majority of specimens collected in the field. We used multiplex polymerase chain reaction for the molecular identification of seven species of wolf spiders that occur commonly in Brassica crops in South Australia—Hogna crispipes, Hogna kuyani, Lycosa godeffroyi, Trochosa expolita, Venator spenceri, Venatrix pseudospeciosa, and a new species in a new genus (‘Species A’). Species-specific primer pairs were designed according to variations in the cytochrome oxidase subunit I gene sequences among of these spider species. Diagnostic DNA fragments for each of the target species allowed species identification. This method proved to be a powerful tool for the identification of this group of arthropods that is difficult to identify based on morphology.

ASSESSMENT OF PRIMERS DESIGNED FROM THE SMALL RIBOSOMAL SUBUNIT RNA FOR SPECIFIC DISCRIMINATION BETWEEN BABESIA BIGEMINA AND BABESIA BOVIS BY PCR

Six pairs of species-specific primers were designed from the alignment of the sequences of the SS rRNA gene obtained from the Genbank database for Babesia bigemina (accession number X59604) and for B. bovis (accession number U06105). Three pairs of primers were designed specifically for B. bigemina and another 3 sets of primers for B. bovis. All oligonucleotide sequences selected as primers were examined for similarities to other organisms through the Genbank Blast procedure and these 6 sets of primers demonstrated high level of specificity. The synthetic oligonucleotides were also tested for specificity by PCR assay using genomic DNA extracted from 40 isolates of B. bigemina and 30 from B. bovis, obtained in six different States of Brazil. All 6 sets of primers were validated as 100% specific for the respective parasite. The PCR amplified the expected fragments for each set of primers, as follows: a) B. bigemina: primers GAU5 forward/GAU6 reverse with amplicon of 1,124 bp; primers GAU5 forward/GAU8 reverse with amplicon of 458 bp; primers GAU7 forward/GAU6 reverse with amplicon of 685 bp; b) B. bovis: primers GAU9 forward/GAU10 reverse with amplicon of 541 bp; primers GAU9 forward/GAU 13 reverse with amplicon of 883 bp; primers SOGIN forward/GAU 10 with amplicon of 1211 bp. KEYWORDS: Babesia bovis, Babesia bigemina, SS rRNA gene, PCR, primers, bovine babesiosis.

Identification of Lactobacillus spp. in probiotic products by real-time PCR and melting curve analysis

A rapid and reliable technique for identifying the strains of lactic acid bacteria (LAB) was developed in this study, and the 16S ribosomal RNA gene (16S rRNA gene) was used as the target. Several species-specific primer pairs were designed based on the variability of 16S rRNA sequence(s) for differentiating 5 strains of lactobacilli which were added into probiotic products in Taiwan. It was simple to identify Lactobacillus acidophilus and L. delbrueckii by species-specific primers, but it could not be used to distinguish L. casei, L. paracasei and L. rhamnosus. Another PCR approach was developed with hybridization probes which were designed according to the difference among the 16S rRNA genes of L. casei, L. paracasei and L. rhamnosus, and melting curve analysis of the hybridization probe was used to distinguish them. It was found that this approach could identify L. paracasei and L. rhamnosus correctly but not separate L. paracasei from L. casei, the result was due to both of them had the same 16S rRNA sequence. These results suggest that melting curve analysis of PCR approach in this study is a rapid, simple and accurate method in distinguishing the closely related strains of lactobacilli.

Molecular identification of vaginal lactobacilli isolated from Bulgarian women

Lactobacilli play an important role in maintaining the vaginal health of women. The development of suitable bacterial replacement therapies for the treatment of vaginosis requires knowledge of the vaginal lactobacilli species representation. The aim of this study was to identify at the species level vaginal Lactobacillus isolates obtained from Bulgarian women in childbearing age by using different molecular methods. Twenty-two strains of lactobacilli isolated from vaginal samples were identified and grouped according to their genetic relatedness. A combined approach, which included amplified ribosomal DNA restriction analysis (ARDRA), ribotyping and polymerase chain reaction (PCR) with species-specific oligonucleotide primers was applied. All vaginal isolates were grouped into 5 clusters in comparison with a set of 21 reference strains based on the initial ARDRA results, which was then confirmed by ribotyping. Finally, the strains were subjected to PCR analysis with eight different species-specific primer pairs, which allowed most of them to be classified as belonging to one of the following species: Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus and Lactobacillus plantarum. In conclusion, this study suggests that the most straightforward identification strategy for vaginal lactobacilli would be grouping by ARDRA or ribotyping, followed by PCR specific primers identification at species level.

Occurrence, Distribution, and Polymerase Chain Reaction-Based Detection of Resistance to Sterol Demethylation Inhibitor Fungicides in Populations of Blumeriella jaapii in Michigan

ABSTRACT The intensive use of site-specific fungicides in agricultural production provides a potent selective mechanism for increasing the frequency of fungicide-resistant isolates in pathogen populations. Practical resistance occurs when the frequency and levels of resistance are great enough to limit the effectiveness of disease control in the field. Cherry leaf spot (CLS), caused by the fungus Blumeriella jaapii, is a major disease of cherry trees in the Great Lakes region. The site-specific sterol demethylation inhibitor fungicides (DMIs) have been used extensively in the region. In 2002, CLS control failed in a Michigan orchard that had used the DMI fenbuconazole exclusively for 8 years. That control failure and our observations from around the state suggested that practical resistance had developed in B. jaapii. Field trial data covering 1989 to 2005 for the DMIs fenbuconazole and tebuconazole supported observations of reduced efficacy of DMIs for controlling CLS. To verify the occurrence of fungicide-resistant B. jaapii, monoconidial isolates were collected in two surveys and tested using a fungicide-amended medium. In one survey, 137 isolates from sites with different DMI histories (no known history, mixed or alternated with other fungicides, and exclusive use) were tested against 12 concentrations of fenbuconazole, tebuconazole, myclobutanil, and fenarimol. Isolates from sites with no prior DMI use were DMI sensitive (DMI(S) = no colony growth at 0.2 mug/ml a.i.) whereas the isolates from the site with prior exclusive use showed growth at DMI concentrations 3 to >100 times higher, and were rated as DMI resistant (DMI(R)). A second survey examined 1,530 monoconidial isolates, including 1,143 from 62 orchard sites in Michigan, where DMIs had been used to control CLS. Resistance to fenbuconazole was detected in 99.7% of the orchard isolates. All isolates from wild cherry trees were sensitive and isolates from feral and dooryard trees showed a range of sensitivities. A polymerase chain reaction (PCR)-based detection method for identifying B. jaapii and DMI(R) was developed and tested. The species-specific primer pair (Bj-F and Bj-R) based on introns in the CYP51 gene of B. jaapii, and the DMI(R)-specific primer pair (DMI-R-Bj-F and DMI-R-Bj-R) based on an insert found upstream of CYP51 in all DMI(R) isolates, provided an accurate and rapid method for detecting DMI(R) B. jaapii. The PCR-based identification method will facilitate timely decision making and continued monitoring of DMI(R) subpopulations in response to management programs.