Internal Transcribed Spacer Polymerase Chain Reaction Research Articles

Antifungal activity of lactic acid bacteria isolated from raw camel milk againts Alternaria alternata and identification of antifungal substance

The phytopathogenic filamentous fungi are responsible for the deterioration of various food products, such as fruits or vegetables, causing significant economic losses. They are also capable of producing several mycotoxins in infected plants and fruits.Lactic acid bacteria (LAB) are considered to be natural antagonists of these dangerous microorganisms. In the present study, 16 strains of LAB isolated from raw camel milk from various regions of Algeria, were the subject of phenotypic and genotypic identification, and they were tested for their antifungal activity. The well method was performed on five strains of lactic acid bacteria against four strains of the toxigenic and spoilage phytopathogenic fungus, Alternaria alternata, isolated from the stems, leaves, roots and fruits of tomatoes and carrots. A study of nature metabolites showed that these metabolites are stable at temperature, and keep the nature of the metabolites with the treatment with proteolytic enzymes such as Pepsin, Lysozyme and Trypsin, so their activity increases with increasing the pH of the culture medium. No loss of antifungal activity of the five selected strains was observed after treatment of the metabolites by heating at 4°C, 30°C, 45°C and 90°C. Internal transcribed spacer polymerase chain reaction (ITS-PCR) analysis and 16S rDNA sequencing were used to characterize and identify LAB isolates that showed varying levels of inhibition of fungal growth. Two strains of LAB, identified as Enterococcus lactis and E. faecium, had the strongest antifungal activity, suggesting potential application in food technology as bio-preservatives against phytopathogenic and food-altering fungi.

Selection and screening of fungal endophytes against wheat pathogens

Tissues of field grown winter wheat ‘AC Morley’ and ‘25R34′ and spring wheat ‘Sumai’ and ‘Scotia’ were collected from Ariss, Ontario in June 2016. These cultivars are considered to be moderately resistant to Fusarium head blight. More than 90 endophytic fungal isolates with 54 distinct morphotypes were obtained from these tissues. These fungal isolates were screened using dual culture plates for antagonism against three wheat pathogens: Fusarium graminearum, Waitea circinata, and Microdochium majus. Out of 54 isolates, 33 were found to cause obvious inhibition of pathogen growth or demarcation lines with at least one of the three wheat pathogens. These antagonistic strains were then tested for pathogenicity on wheat leaves in growth room tests, and 16 showed no phytopathogenicity. Among these non-pathogenic strains, three were selected and identified by internal transcribed spacer-polymerase chain reaction (ITS-PCR) as Valsa friesii, Simplicillium lamellicola and Cladorrhinum flexuosum. These three were then tested for their ability to inhibit disease in growth room tests. Hyphal plugs of these fungi inhibited the pathogenicity of wheat pathogens F. graminearum, and W. circinata on leaves of wheat seedlings, and reduced lesion lengths by 36–87% and 31%-86%, respectively. The wheat endophytes, S. lamellicola and C. flexuosum, are promising biocontrol agents against F. graminearum and W. circinata.

Molecular identification of bovine trypanosomes in relation to cattle sources in southwest Nigeria

Bovine trypanosomosis is a problem in the livestock industry in Nigeria. A longitudinal survey of cattle sampled during the wet and dry seasons was conducted from April 2016 to March 2017. Blood samples were collected by random sampling from 745 cattle in southwest Nigeria and screened for trypanosomes by internal transcribed spacer–polymerase chain reaction (ITS-PCR). Cattle positive for Trypanozoon DNA were further screened with the Rode Trypanozoon antigen type (RoTat) 1.2 PCR and Trypanosoma brucei gambiense glycoprotein (TgsGP) genes for T. evansi and T. b. gambiense respectively. Trypanosome DNA was amplified in 23.8% (95%CI: 20.8–26.9) of cattle with significantly higher prevalence in wet season (95%CI: 22.9–30.8) when compared to the dry season (95%CI: 14.3–23.6). A high prevalence was observed in Fulani cattle farms 54.1% (95%CI: 42.78–64.93%) while the prevalence was lower in institutional farms 14.7% (95%CI: 10.10–20.97%). Trypanosoma vivax was the most prevalent trypanosome observed (11.54% (95%CI: 9.44–14.04%)), followed by T. congolense 8.5% (95%CI: 6.67–10.67%) T. b. brucei 4.8% (95%CI: 3.51–6.62%) and T. evansi 1.74% (95%CI: 1.02–2.96%). Mixed infections were observed in 2.8% (95%CI: 1.85–4.27%) of cattle. Seasonal variation revealed a predominance of T. congolense and T. vivax in wet and dry season, respectively. The high prevalence of Trypanosoma species in cattle indicates a need for expanded surveillance for AAT in southwest Nigeria. Migration, settlement patterns, increased marketing and management types were some of the risk factors identified for AAT.

Isolation and screening of lipolytic fungi from coastal waters of the southern caspian sea (north of iran).

Background:Lipases (acylglycerol acylhydrolase, E. C. 3. 1. 1. 3) are widely distributed among microorganisms, animals and plants, catalyzing the hydrolysis of triglycerides to free fatty acids and glycerol. Their commercial application includes pharmaceutical, chemical, and paper industries.Objectives:This study aimed to isolate and screen lipolytic fungi from coastal waters of the southern Caspian Sea by Internal Transcribed Spacer-Polymerase Chain Reaction (ITS-PCR), and to optimize their lipolytic activity, pH and temperature. The ITS regions possess a high variation among taxonomically distinct fungal species and even within species.Materials and Methods:All fungal were tested to determine their lipolytic activity by the Tributyrin agar plate assay. After DNA extraction, lipase-producing fungi were identified via ITS-PCR of rDNA region with ITS1 and ITS4 primers.Results:Four fungal species were isolated from water samples of the Caspian Sea (north of Iran) between February and June 2011. The nucleotide sequences reported for three of these isolates have been assigned accession numbers from NCBI Gene Bank database. Among these species, Cladosporium langeronii showed maximum lipolytic activity (34 U/mL) and maximum clear zone formation (6 mm) on the Tributyrin agar plates. The optimum pH and temperature for activity were 8.0 and 35°C, respectively.Conclusions:The findings of this study indicated that these isolates were plant pathogenic fungi, which entered seawater from the environment, and were likely to have a suitable lipase activity on plant oils.

English

The objectives of this research were to characterize isolates of Trichoderma collected from rhizospheres of chickpea, pigeonpea and lentil crop from different places of Uttar Pradesh, India, using microsatellite-primed polymerase chain reaction (MP-PCR) and ribosomal DNA (rDNA) sequence analysis and to combine these results with morphological characteristics for clas­sification. Thirty isolates of Trichoderma sp. obtained from rhizosphere soil of plantation crops, and agricultural fields of UP region were studied using inter-simple sequence repeat (ISSR) and Internal transcribed spacer- polymerase chain reaction (ITS-PCR). The genetic relatedness among 15 isolates of Trichoderma sp. was analyzed with six micro-satellite primers. ISSR profiles showed 83.7% genetic diversity among the isolates with the formation of four clusters. Analysis of dendrogram revealed that similarity coefficient ranged from 0.27 to 0.95. ITS-PCR of rDNA region with ITS1 and ITS4 primers produced 600 bp products in all isolates. This result presented the identification patterns of Trichoderma isolates.   Key words: Trichoderma sp., genetic diversity, polymerase chain reaction (PCR), molecular marker, microsatellite.

Methicillin-resistant Staphylococcus sp. colonizing health care workers of a cancer hospital.

The aim of the study was to analyze epidemiological and microbiological aspects of oral colonization by methicillin-resistant Staphylococcus of health care workers in a cancer hospital. Interview and saliva sampling were performed with 149 health care workers. Antimicrobial resistance was determined by disk diffusion and minimum inhibitory concentration. Polymerase Chain Reaction, Internal Transcribed Spacer-Polymerase Chain Reaction and Pulsed Field Gel Electrophoresis were performed for genotypic characterization of methicillin-resistant Staphylococcus. Risk factors were determined by logistic regression. Methicillin-resistant Staphylococcus colonization prevalence was 19.5%, denture wearing (p = 0.03), habit of nail biting (p = 0.04) and preparation and administration of antimicrobial (p = 0.04) were risk factors identified. All methicillin-resistant Staphylococcus were S. epidermidis, 94.4% of them had mecA gene. Closely related and indistinguishable methicillin-resistant S. epidermidis were detected. These results highlight that HCWs which have contact with patient at high risk for developing infections were identified as colonized by MRSE in the oral cavity, reinforcing this cavity as a reservoir of these bacteria and the risk to themselves and patients safety, because these microorganisms may be spread by coughing and talking.

Genome Subtyping of Autochthonous Bacillus Species Isolated from Iru, a Fermented Parkia biglobosa Seed

Phylogenetic relationship and strains sub-typing of Bacillus species isolated from iru, a traditional fermented condiment in Africa were studied using polyphasic genomic approaches and the profiles compared with bacilli isolated from similar Asian condiments. The 16S rRNA gene sequencing identified the strains as Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus licheniformis, Bacillus pumilus, and Brevibacillus formosus. The phylogenetic analysis conducted showed five distinct clusters with genetic relatedness among B. subtilis and B. amyloliquefaciens strains from Africa and Asia. Amplified ribosomal DNA restriction analysis (ARDRA) successfully differentiated species of B. subtilis phylogeny from B. cereus. Combined analyses of ARDRA, internal transcribed spacer-polymerase chain reaction (ITS-PCR), ITS-PCR-restriction fragment length polymorphism (ITS-PCR-RFLP) and randomly amplified polymorphic DNA (RAPD-PCR) further confirmed B. subtilis and B. amyloliquefaciens as the dominant Bacillus species associated with fermentation of iru, and revealed high strains genetic diversity, while multilocus sequence analysis (MLSA) data distinguished B. cereus from B. thuringiensis. This information is essential for selection of starter cultures with desirable functional attributes to guarantee product consistency and safety quality of traditional fermented foods.

Species-Specific PCR Assays for Differentiating Heterodera filipjevi and H. avenae.

Heterodera avenae and H. filipjevi are economically important cyst nematodes that restrict production of cereal crops in the Pacific Northwest United States and elsewhere in the world. Identification of these two species is critical for recommending and implementing effective management practices. Primers were designed from the internal transcribed spacer (ITS) regions of H. avenae and H. filipjevi ribosomal DNA. The primers were highly specific when examined on target isolates but did not amplify DNA from nontarget Heterodera, Globodera, Meloidogyne, Pratylenchus, and other nematode species tested. Polymerase chain reaction (PCR) and amplification conditions were established, and H. avenae and H. filipjevi were clearly distinguished by PCR fragments of 242 and 170 bp, respectively. Robust PCR amplification was achieved with DNA extracted from a single egg or second-stage juvenile (J2) using a laboratory-made worm lysis buffer, and DNA from 0.5 egg or J2 using a commercial kit. The PCR assays were successfully employed for differentiation of H. filipjevi and H. avenae populations collected from eight locations in three Pacific Northwest states. This is the first report of a species-specific ITS PCR assay to detect and identify H. filipjevi. The assays for both species will enhance diagnosis of cereal cyst nematode species in infested fields.

Molecular prevalence and preponderance of Eimeria spp. among chickens in Tamil Nadu, India

Coccidosis is one of the most commonly prevalent and economically important parasitic diseases of poultry worldwide. Chicken coccidia are protozoan parasites of the genus Eimeria. This study aimed at analysing the molecular prevalence of seven species of Eimeria infecting chickens in Tamil Nadu, India. Tissue samples (caecum, rectum and upper and mid intestines) collected from chickens exhibiting symptoms of coccidiosis were used for DNA extraction, followed by amplification of the internal transcribed spacer (ITS) region of Eimeria genome with genus-specific primers and speciation in nested polymerase chain reaction (PCR) with species-specific primers. Of 43 tissue samples examined, 25 were positive in ITS PCR and all the seven species could be identified. However, the prevalence of each species varied. In broilers, Eimeria necatrix was present in all infected chickens with Eimeria brunetti, Eimeria tenella, Eimeria maxima and Eimeria acervulina present in more than 50% of infected chickens, while Eimeria praecox and Eimeria mitis were only present in 11% to 16%. Although only 7 samples were positive among layers, the prevalence was largely similar, but with a higher prevalence of E. praecox and E. mitis and a lower prevalence of E. tenella. Multiple infections were most common, with 2-6 Eimeria species infecting the same chickens. In order to estimate the preponderance of each infecting species of Eimeria, a random cloning technique was adopted. The genus-specific ITS PCR product was cloned in a TA vector and ten clones were randomly picked and used as template for amplification of all the seven genera of Eimeria. If the specific species of Eimeria is preponderant, then the frequency of the clones showing that species-specific PCR amplification would be higher. Using this method, the most preponderant species present in the rectum, mid and upper intestines of layers was assessed to be E. acervulina, E. brunetti and E. necatrix. E. acervulina was present in 60-90%, E. necatrix in 10-30% and E. brunetti in 10-20% of the clones screened, indicating that these species could be the most preponderant Eimeria species. Intervention strategies should aim at these species. This new method of estimating preponderance of infecting Eimeria species could be used to assess the relative importance of each species at the farm or region level instead of relying only on prevalence estimates.

Localised Microsphaeropsis Arundinis Infection of the Subcutis of a Cat

CLINICAL PRESENTATION AND DIAGNOSIS: An 11-year-old spayed domestic crossbred cat was presented for two focal lesions affecting the subcutis that developed 3 weeks after a cat fight. Lesions consisted of swelling of the subcutaneous tissues over the bridge of the nose and a focal swelling of the dewclaw pad of the left thoracic limb. Both lesions were attributable to localised infections with the unusual fungal pathogen Microsphaeropsis arundinis, based on direct microscopy of smear cytology, histology, fungal culture, internal transcribed spacer-polymerase chain reaction (ITS-PCR) and sequence analysis of formalin-fixed tissue and colonial material from primary culture. The dewclaw pad lesion resolved following cytoreductive surgery and a course of itraconazole (50 mg once daily). The nasal bridge lesion was treated in a similar fashion, except that a range of different antifungal agents (itraconazole, fluconazole, posaconazole) were used, administered sequentially. Despite this, the infection recurred on three subsequent occasions, and the cat has been prescribed indefinite azole therapy. This report of a refractory localised infection of the subcutis in an apparently immune-competent cat illustrates the value of using a mycology reference laboratory, and PCR and sequence analysis for organism identification, and the therapeutic potential of new antifungal agents such as posaconazole.

Retrospective analysis of the genetic diversity of Klebsiella oxytoca isolated in Poland over a 50-year period

Population genetics analyses and determination of the phylogenetic relationships between strains have proven to be extremely useful approaches, enabling deeper insights into the epidemiological pattern of bacterial species. There is no longitudinal data describing the molecular epidemiology of Klebsiella oxytoca strains that are opportunistic pathogens responsible for an increasing number of multi-resistant infections in hospitals. The aim of the present study was to assess the genetic diversity of K. oxytoca strains over a 50-year period using internal transcribed spacer polymerase chain reaction (ITS-PCR) and PCR MP (ang. PCR melting profiles) genotyping methods on a large collection of strains isolated from the patients of several hospitals in Poland. The phylogenetic analysis based on ITS-PCR exhibited six distinct branches. Two main groups, KoX and KoY, with four and two sub-groups within KoX and KoY, respectively, have been identified. Typing by the PCR MP method showed a higher level of genetic diversity. However, all K. oxytoca strains were also divided into six genotype groups (KoA, KoB, KoC, KoD, KoE and KoF). In conclusion, we found that the ITS-PCR and PCR MP methods are useful for the phylogenetic delineation of genetic groups in K. oxytoca.

Perpetuation of Powdery Mildew Infection and Identification of Erysiphe australiana as the Crape Myrtle Pathogen in Mid-Tennessee.

The fungus Erysiphe lagerstroemiae is commonly known as the powdery mildew pathogen in crape myrtle (Lagerstroemiae indica) in the United States, and Erysiphe australiana is the powdery mildew pathogen reported in Japan, China, and Australia. The teleomorph often used to identify powdery mildew fungi rarely develops in crape myrtle, and in our observations, ascocarps never formed. Our study showed that the crape myrtle pathogen overwintered as mycelia on dormant buds. The internal transcribed spacer (ITS) regions of rDNA and the intervening 5.8S rRNA gene were amplified using standard polymerase chain reaction (PCR) protocols and the universal primer pairs ITS1 and ITS4. PCR products were analyzed by electrophoresis in a 1.5% agarose gel and sequenced, and the ITS PCR product was 666 bp from ITS1/ITS4 and 704 bp from ITS1-F/ITS4. BLAST analysis of the sequence of the PCR products showed identical similarity with E. australiana reported in Japan, China, and Australia. Comparison of ITS sequences with information in the GenBank on other powdery mildew fungi showed a closest alignment (93% similarity) to Erysiphe juglandis that infects walnut. Specific primers for E. australiana were developed and evaluated for use as diagnostic tools. Out of 12 specific primer pairs evaluated, four primer pairs and four double primer pairs were highly specific to E. australiana and did not amplify Erysiphe pulchra of dogwood, Erysiphe syringae of common lilac, Erysiphe circinata of maple, or Phyllactinia guttata of oak. The E. australiana-specific primers amplified 16 samples of crape myrtle powdery mildew collected from diverse locations in mid-Tennessee. These results clearly showed that the crape myrtle powdery mildew in mid-Tennessee was caused by E. australiana. Specific primers reported in this article provide a diagnostic tool and may be used to confirm the identity of crape myrtle powdery mildew pathogen in other areas in the United States and wherever the disease occurs.

QUANTIFYING DISPERSAL AND ESTABLISHMENT LIMITATION IN A POPULATION OF AN EPIPHYTIC LICHEN

Dispersal is a process critical for the dynamics and persistence of metapopulations, but it is difficult to quantify. It has been suggested that the old-forest lichen Lobaria pulmonaria is limited by insufficient dispersal ability. We analyzed 240 DNA extracts derived from snow samples by a L. pulmonaria-specific real-time PCR (polymerase chain reaction) assay of the ITS (internal transcribed spacer) region allowing for the discrimination among propagules originating from a single, isolated source tree or propagules originating from other locations. Samples that were detected as positives by real-time PCR were additionally genotyped for five L. pulmonaria microsatellite loci. Both molecular approaches demonstrated substantial dispersal from other than local sources. In a landscape approach, we additionally analyzed 240 snow samples with real-time PCR of ITS and detected propagules not only in forests where L. pulmonaria was present, but also in large unforested pasture areas and in forest patches where L. pulmonaria was not found. Monitoring of soredia of L. pulmonaria transplanted to maple bark after two vegetation periods showed high variance in growth among forest stands, but no significant differences among different transplantation treatments. Hence, it is probably not dispersal limitation that hinders colonization in the old-forest lichen L. pulmonaria, but ecological constraints at the stand level that can result in establishment limitation. Our study exemplifies that care has to be taken to adequately separate the effects of dispersal limitation from a limitation of establishment.

Anticlostridial activity of Lactobacillus isolated from semi-hard cheeses

Non-starter lactic acid bacteria grow to high numbers in semi-hard cheeses during ripening, and may suppress harmful bacteria. In this study, about 400 Lactobacillus isolates from Danish semi-hard cheeses were identified to species level using internal transcribed spacer-polymerase chain reaction (ITS-PCR) analysis. The majority of isolates belonged to the Lb. paracasei complex and were classified into approximately 135 types using pulsed field gel electrophoresis (PFGE). Lactobacillus isolates representing all the different PFGE types were screened, using an agar well diffusion assay, for antimicrobial activity against 15 single-strain Clostridium cultures. Almost half of the isolates possessed anticlostridial activity, and 10% possessed a broad and consistent activity. Nine strains were further investigated for properties of importance for use as mixed cultures in cheese and silage. The results showed that anticlostridial non-starter Lactobacillus growing in high quality semi-hard cheeses could be useful as protective adjunct cultures against the growth of Clostridium .

Evaluation of a species-specific PCR assay for the Anopheles funestus group from eleven African countries and Madagascar

A newly published cocktail polymerase chain reaction (PCR) assay can identify five members of the Anopheles funestus group: An. funestus, An. vaneedeni, An. parensis, An. leesoni and An. rivulorum. The assay was evaluated on specimens from 11 African countries: Angola, Cote d’Ivoire, Ethiopia, Kenya, Malawi, Mozambique, Namibia, South Africa, Tanzania, Uganda and Zambia; and the island of Madagascar. The polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) and the internal transcribed spacer PCR (ITS2-PCR) assays were used as a priori identification methods on 900 specimens. Of these, 96.4% were correctly identified using the new cocktail PCR method. The remaining 3.5% (32) from Malawi failed to amplify. The failure to identify these samples was not significantly different from the samples from the other countries (Chi-square; P>0.05). The results suggest that the new species-specific PCR assay is an efficient and effective means of identifying 5 members of the An. funestus group in a single reaction. It is also less time-consuming than the molecular methods previously used on the group.

Elevated genetic heterogeneity and Pleistocene climatic instability: inferences from nrDNA in New Zealand Coprosma (Rubiaceae)

AimTo examine patterns of hybridization and genotype mixing within the genus Coprosma J.R.Forst. & G.Forst. (Rubiaceae).LocationNew ZealandMethodsNucleotide sequence was determined for the internal transcribed spacer (ITS) and external transcribed spacer (ETS) regions of nuclear ribosomal DNA for fifty individuals from thirty‐six taxa within the New Zealand component of the genus Coprosma.ResultsMixed sequences were found to be widespread in Coprosma. Direct sequencing of ITS polymerase chain reaction (PCR) products from seven polyploid taxa showed evidence of sequence mixtures. Cloning and sequencing of individual PCR products from two polyploids confirmed the presence of multiple templates, one of which corresponded to that of a diploid. Intra‐individual heterogeneity was also seen in a hybrid diploid taxon, with the mixed nucleotides corresponding to those of the parental lineages. Finally the ITS sequences of twenty‐two diploid taxa showed that eleven contained intra‐individual heterogeneity.ConclusionsWe conclude that the widespread occurrence of sequence mixtures in Coprosma results from of frequent hybridization. We also conclude that concerted evolution of the ITS and ETS regions is depressed. We propose that these characteristics evolved as a mechanism to maintain high levels of heterogeneity and suggest that this is adaptive for Coprosma in climatically unstable and physically complex New Zealand landscapes. These landscapes have been subjected to repeated oscillations between stadial and interstadial environments during the Pleistocene.

Improvement of the identification of staphylococci isolated from bovine mammary infections using molecular methods

Fifty-six Staphylococcus strains isolated from cases of bovine mammary infections were identified by using phenotypic and genotypic methods. Twenty-eight strains (50%) were identified at the species level according to their phenotypic characteristics, whereas the remaining 28 strains presented atypical or unreliable profiles. A combination of phenotypic and genotypic methods allowed the 56 strains studied to be classified. Internal transcribed spacer-polymerase chain reaction (ITS-PCR) based on the polymorphism of the 16S-23S rDNA spacer region appeared as a rapid and reliable method for the classification of bovine staphylococcal isolates at the species and subspecies levels.

Molecular and morphological discrimination betweenTylospora fibrillosaandTylospora asterophoramycorrhizae

Among the mycorrhizal types of spruce, Tylospora-type mycorrhizae are the most constant and abundant. Two species of the genus Tylospora occur in Europe, Tylospora fibrillosa and Tylospora asterophora. Mycorrhizae of T. asterophora are described in detail for the first time. Sequences of the internal transcribed spacer (ITS) of the ribosomal genes were obtained from T. fibrillosa and T. asterophora mycorrhizae, sporocarps, and cultured mycelium. Discrimination and identification of the two species by ITS polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) are discussed in the light of inter- and intra-specific variability. Species-specific PCR primers were designed to distinguish both species. Molecular screening of Tylospora-type mycorrhizae from field material led to unambiguous results, whereas morphological identification is likely to fail because of great similarity even at the microscopic level.Key words: Tylospora asterophora, Tylospora fibrillosa, ectomycorrhizae, taxon specific primers (TSOPs), ITS sequences.

Fruit fly (Diptera: Tephritidae) species identification: a rapid molecular diagnostic technique for quarantine application

AbstractNew Zealand is currently the only major fruit producing country in the world that is free of economically important fruit flies. As part of the effort to maintain this status, there is a need to supplement quarantine decision-making procedures with a means of rapidly identifying immature life stage infestations to the species level. Here we describe a molecular method that achieves this, using simple restriction patterns of ribosomal DNA (rDNA) as diagnostic markers. The 18S and 18S plus internal transcribed spacer (ITS) regions were amplified from larval DNA by the polymerase chain reaction (PCR). Nineteen species, spanning four genera (including five subgenera ofBactrocera) were analysed. Restriction analysis of the 18S PCR product provided poor resolution, even at the generic level. Digestion of the 18S + ITS PCR product, however, generated thirteen diagnostic haplotypes as defined by the composite restriction patterns fromRsaI,Sau3aHaeIII andAluI. No variation was detected at these restriction sites within or between populations. Twenty two restriction enzymes have been screened, but diagnostic RFLPs have yet to been found for six out of the tenBactrocera (Bactrocera)species;B. passiflorae(Froggatt) cannot be distinguished fromB. facialis(Coquillet), norB. kirki(Froggatt) fromB. trilineola(Froggatt) orB. neohumeralis(Hardy) fromB. tryoni(Froggatt). Geographic origin could assist in distinguishing the first four species, but the latter pair are very closely related with overlapping origins, hosts and adult morphology. All six species, however, are considered high risk with respect to their likely establishment in New Zealand. Therefore diagnosis based on this molecular technique would support the same quarantine decision. We consider this method could be useful as a diagnostic technique and discuss directions for further development.