Species-specific Primer Pairs Research Articles

Full-genome characterization of Turkish Rose Yellow Vein Virus (RYVV) Isolates

Rose yellow vein virus (RYVV) is a viral agent that has been identified in roses in recent years and is known to exist in a very limited area in the world. However, in studies conducted in our country, the presence of the agent has been detected in different regions and provinces. Since direct chemical control of viral diseases is not possible as in other biotic diseases, molecular characterization of the agent forms the basis for possible resistance or gene silencing studies to be conducted in the coming years. Thus, 44 RYVV isolates previously identified in our country were used for full genome studies. In full genome studies, overlapping PCR method was used with 7 different species-specific primer pairs. The full genomes of 3 of these isolates were obtained. The full genomes of the other isolates could not be obtained. It was determined that the genome size of the 3 RYVV isolates whose full genomes were obtained was 9314 nucleotides. It was determined that the only RYVV isolate found in the GenBank and the Türkiye isolates also showed a high level of nucleotide sequence similarity. However, the fact that the entire genome of the other isolates could not be obtained and the ones obtained were amplified with primer pairs designed according to the single isolate in the GenBank database may be due to the presence of different strains of RYVV isolates. For this reason, it is thought that next-generation sequencing studies should be prioritized to obtain complete genome information in future studies.

Molecular authentication and discrimination between banana shrimp and Pacific white shrimp by duplex PCR-lateral flow dipstick (dPCR-LFD) system

Banana shrimp is easily substituted by Pacific white shrimp due to its relatively similar morphology and appearance. The duplex PCR-lateral flow dipstick (dPCR-LFD) system was developed to authenticate these two species based on the mitochondrial cytochrome b (Cytb) gene. Two pairs of species-specific primers were designed for dPCR amplification. The specificity was also tested toward nine marine species. The sensitivity of the method was also evaluated at the DNA range of 0.02 %-60 % of adulterated Pacific white shrimp DNA in DNA from banana shrimp. Several types of cooked samples under various conditions were also tested. The developed dPCR-LFD system could specifically differentiate banana shrimp from Pacific white shrimp. No cross-amplification was observed in the specificity test. The sensitivity test showed the detection limit of 0.02 % (0.01 ng) of Pacific white shrimp DNA. The cooking processes including boiling, frying, and steaming did not show any effect on the efficiency of the dPCR-LFD system. This technique could be therefore implemented for the authentication of banana shrimp and Pacific white shrimp products as well as further use for other seafood frauds.

First Report of Meloidogyne enterolobii Infecting Solanum nigrum in China.

Black nightshade (Solanum nigrum) typically grows as a weed species, but it is also widely used as an herb to treat stomach ulcers and dermal infections in many countries (Jabamalairaj et al. 2019). In April 2023, extensive root galls similar to those associated with by root-knot nematodes (RKNs), Meloidogyne spp., were observed on the roots of black nightshade in several commercial fields in Lufeng county (22°55'57.44″N, 115°33'10.31″E), Guangdong Province, China. Upon inspection, there were one to several female RKN in each gall, and egg masses protruding through the root surface. The disease incidence rate was more than 90% in each field using the random sampling method. The nematode population densities in the samples ranged from 279 to 656 eggs and second-stage juveniles (J2s) per gram of fresh roots. Females and egg masses were collected from the roots, and egg masses were incubated in sterile water at 25°C to obtain J2s. Males were not collected in root galling or soil samples. The J2 tail is thin with a broad, bluntly pointed tip, and a clearly defined hyaline tail terminus. Measurements of J2 (n = 20) included: L= 440 ± 30.5 (384 to 500) µm, stylet = 12.3 ± 0.7 (11.3 to 13.7) µm, tail = 51.6 ± 2.4 (47.9 to 57.0) µm. For females (n = 15), vulval slit length = 25.5 ±1.9 (23.6 to 29.1) µm, vulval slit to anus distance = 22.1 ± 3.0 (18.2 to 27.0) µm. Stylet knobs in females are divided longitudinally by a groove so that each knob appears as two. The perineal patterns are round to ovoid, with coarse and smooth striae, moderate to high dorsal arch and mostly lacking distinct lateral lines. Morphological characteristics from J2s and perineal patterns from adult females fit the original description of M. enterolobii (Yang and Eisenback 1983). Furthermore, species identity was explored by sequencing the D2-D3 region of the 28S rRNA gene using primers D2A/D3B (Vrain et al. 1992), and the mtDNA cytochrome c oxidase I (COI) genes using primers JB3/JB5 (Derycke et al. 2005). The sequences for the target genes were 759 bp (GenBank Accession No. OR046056) and 447 bp (GenBank Accession No. OR042802), respectively. The BLAST analysis suggested 98.17~99.78% similarities to other available M. enterolobii sequences in GenBank. Species identity was further confirmed with the species-specific primer pair Me-F/Me-R (Long et al. 2006). An approximately 240 bp PCR product was produced, which was previously reported only for M. enterolobii, whereas no product was obtained from control populations of M. incognita or M. javanica. The pathogenicity test was conducted in a greenhouse at 28°C using seedlings of S. nigrum maintained in pots containing 500 cm3 sterilized soil. Ten replicates were inoculated with 800 eggs and J2s of the original population of M. enterolobii, while another 10 replicates of control plants were not inoculated. After 7 weeks, the inoculated plants exhibited galling symptoms similar to plants observed in the field, and females and egg masses were obtained by dissecting galls. No galling symptoms were observed on control plants. These results confirmed the nematode's pathogenicity. To our knowledge, this is the first record of M. enterolobii parasitizing black nightshade. M. enterolobii stands out as a highly deleterious variant among the species of RKNs owing to its extensive repertoire of host plants, pathogenicity, and proficiency in thriving and multiplying even on crops possessing resistance genes (Sikandar, 2022). In addition to being a medicinal plant, S. nigrum is a widespread weed found in fields throughout China. This report also showed that S. nigrum could play an important role as a reservoir host of M. enterolobii aiding its survival, reproduction, spread, and increasing the potential damage for host crops.

First report of Pseudomonas mediterranea causing tomato pith necrosis in Croatia.

In May 2021, a tomato producer reported an occurrence of a disease unknown so far in a greenhouse near Split, Croatia. About 30% of plants (cultivar Signora) have been affected. Symptoms resembled tomato pith necrosis, bacterial disease caused by Pseudomonas corrugata, known to occur sporadically in tomato greenhouse production in Croatia. Leaves on plants developed interveinal chlorosis, followed by necrosis and leaf collapse. When main stems were longitudinally cut, brown, disintegrated and water-soaked partly hollow pith was evident. Severely affected plants wilted. With suspicion on presence of P. corrugata, bacteria were isolated from surface-sterilized pith tissue of two tomato plants by plating onto sucrose peptone agar (SPA) and King's B medium (KB). Colonies recovered were cream-colored on SPA and non-florescent on KB. Two isolates, assigned as 1-KB and 3A, were first identified by amplification of internal transcribed spacer (ITS1) between16S rRNA and 23S rRNA using primers D21 and D22 (Manceau and Horvais 1997). The 550-bp PCR products obtained were purified and sequenced. Subsequent BLAST search showed the sequences to have 100% identity with the strain DSM 16733 isolated from tomato in Italy (Accession No. LT629790.1) and 99.77% identity with the strain SM664-12 isolated from tomato in USA (Acc. No. KC405207.1) of Pseudomonas mediterranea from NCBI. ITS sequence for one isolate 3A was deposited in GenBank under the Accession No. OP765279.1. Further identification was performed by using species-specific primers PC1/1-PC1/2 for P. mediterranea (Catara et al. 2000, 2002). Amplification of 600 bp DNA fragment confirmed the identity of isolates 1-KB and 3A as P. mediterranea. For this region sequence of isolate 3A was deposited in GenBank under the Acc. No. OP068273.1. Pathogenicity was assessed on tomato plants (cultivar Moneymaker) grown in pots in bio-chamber. Plants were grown at 25/20 °C 12h/12h dark/light regime until 8-leaves stage (BBCH 18). P. mediterranea isolate 3A was used for the inoculation. Inoculum was prepared from the isolate grown on KB medium for 48 h and suspended in sterile distilled water (concentration of 109 CFU mL-1) by dilution plate counts. Ten plants were inoculated with 10 µl of bacterial suspension injected into the stem with a syringe. Five control plants were inoculated with sterile distilled water. After 40 days of plant growth, symptoms were visible on all plants inoculated with P. mediterranea isolate 3A. Although no wilting was observed and all plants were alive, chlorosis was observed on upper leaves, chlorosis and necrosis on middle leaves, while basal leaves wilted. Longitudinal cross-sections of stems revealed brownish pith tissue with longitudinal watery pits spreading from inoculation points (Fig.S1). Symptoms were not observed on control plants. Bacterium was reisolated from three plants showing the most severe symptoms and proved to be identical to the original using species-specific primer pair PC1/1-PC1/2. To our knowledge, this is the first confirmation of P. mediterranea causing tomato pith necrosis in Croatia. Tomato pith necrosis caused by P. mediterranea may become significant bacterial disease of greenhouse tomato in Croatia.

First report of Alternariaster helianthi causing leaf blight disease of sunflower (Helianthus annus) in Sudan

In Sudan, oilseed crops rank second in production area after cereals, and sunflower (Helianthus annuus) is considered a promising oilseed (Mohammed, 2010). Leaf blight caused by Alternariaster helianthi is a destructive disease of sunflower. During autumn 2018 and 2019 diseased sunflowers with dark brown to black leaves and stem spots were observed in the Blue Nile state, Sudan. The disease incidence in the major rain-fed areas, approximately 690 hectares, was 90 and 88% in 2018 and 2019, respectively. Symptoms on the leaf surface were observed most frequently after flowering. The disease appeared in the form of small scattered brown spots on the leaf lamina. Leaf spots were irregular, circular and 5–10 mm in diameter. Later these spots increased in size and coalesced covering a larger leaf area, with a dark brown margin and yellow halo. Necrotic lesions also appeared on the stem, petioles and sepals (Figs. 1, 2) with spots on younger plants exhibiting a yellow halo (Fig. 3). Infected leaf and stem tissues were surface-sterilised with sodium hypochlorite (1% v/v) for three minutes, then rinsed three times with distilled water and plated on potato dextrose agar. The plated tissue produced dark olivaceous to black colonies after five to seven days. The conidia were solitary, non-beaked, borne on simple unbranched conidiophores, cylindrical to elongate and elliptic (Fig. 4). The conidium colour was subhyaline to very dilute tan, becoming slightly darker in the most mature conidia, the conidia were variable in size from 18.1 to 23.9 μm wide and 86 to 112 μm long, and borne singly on the conidiophores. Based on the conidia and conidiophore morphology as described by Simmons (2007), the pathogen was identified as Alternariaster helianthi. For molecular identification, DNA was extracted from a representative isolate (designated AHE-2) using a DNeasy Plant Mini Kit (Qiagen, USA). PCR using a species-specific primer pair AhN1F/AhN1R (Udayashankar et al., 2012) amplified a 314 bp product which was sequenced for confirmation of A. helianthi. The resulting sequence was deposited in GenBank (Accession No. OM618612). BLASTn analysis showed that the sequence had 99% (289/291 bp) identity with an rDNA ITS sequence previously identified as A. helianthi (MK968068). Pathogenicity was determined by inoculating 30 sunflower seedlings (30 days old). Conidia from a one-week-old culture was applied to the seedlings using a 10 ml of suspension containing 106 conidia/ml. Twenty control plants were sprayed with sterile distilled water using a hand atomiser sprayer. Inoculated and control plants were covered with plastic bags and maintained in greenhouse for 24 hours after inoculation. Symptoms similar to those observed in the field appeared on all 30 inoculated plants after a few days, the percentage leaf coverage with symptoms was 38%. Alternariaster helianthi was re-isolated from the symptomatic plants, the re-isolated culture and spores were similar in morphology to the inoculated isolate. No symptoms were observed in the control plants. To our knowledge, this is the first report of A. helianthi in Sudan. Alternariaster helianthi has been reported in Uganda, Kenya, and South Africa (Kgatle et al., 2018; 2019). In this study, high disease incidences were observed, and significant yield losses may occur (Calvet et al., 2005), therefore, management strategies are needed to help prevent further spread.

Identification, molecular characterization and risk factors of Theileria infection among sheep: a first comprehensive report from North India

The present study was aimed at the identification, molecular characterization, and risk factor assessment of Theileria infection among sheep of Haryana province, north India. A total of 402 blood samples were collected from three different climatic zones of Haryana from March 2020 to September 2021. Light microscopy of blood smears revealed Theileria spp. infection in 47.26% (n = 190), while 60.94% (n = 245) of blood samples were positive using nested PCR. Extensive molecular characterization of Theileria infection using four pairs of species-specific primers indicated the dominance of T. ovis (29.1%) followed by T. lestoquardi (12.69%), T. luwenshuni (5.97%) and T. annulata (1.49%). Mixed infection was detected in 11.69% of cases. Bidirectional sequencing and phylogeny further confirmed the presence of these four Theileria spp. in the investigated area under study. Hematology indicated a significant (p < 0.01) reduction in various haematological indices of animals infected with T. luwenshuni and T. lestoquardi compared to the healthy control group. Risk factors like age, sex, and zone were significantly associated with Theileria infection in sheep. The present investigation depicts the first comprehensive molecular report of ovine Theileria spp., which warrants further study to develop suitable control strategies against these haemoparasitic infections.

Development of the Droplet Digital PCR Method for the Detection and Quantification of Erwinia pyrifoliae.

Black shoot blight disease caused by Erwinia pyrifoliae has serious impacts on quality and yield in pear production in Korea; therefore, rapid and accurate methods for its detection are needed. However, traditional detection methods require a great deal of time and fail to achieve absolute quantification. In the present study, we developed a droplet digital polymerase chain reaction (ddPCR) method for the detection and absolute quantification of E. pyrifoliae using a pair of species-specific primers. The detection range was 103 - 107 copies/ml (DNA templates) and cfu/ml (cell culture templates). This new method exhibited good linearity and repeatability and was validated by absolute quantification of E. pyrifoliae DNA copies from samples of artificially inoculated immature pear fruits. Here, we present the first study of ddPCR assay for the detection and quantification of E. pyrifoliae. This method has potential applications in epidemiology and for the early prediction of black shoot blight outbreaks.

Bacterial spot caused by Xanthomonas vesicatoria devastated tomato production of Khyber Pakhtunkhwa-Pakistan

In Khyber Pakhtunkhwa (KP) province-Pakistan, bacterial spot caused by Xanthomonas campestris pv. vesicatoria (Xcv) has decimated commercial tomato production. Field surveys were conducted in 2017 and 2018 to analyse the effects of the bacterial spot in tomato producing areas of KP, including Swat, Lower Dir, and Mansehra. During the growing seasons, diseased samples were collected from tomato fields, and disease incidence and severity were documented. The pathogen was isolated, purified, and morphologically and biochemically characterised. Most of the morphological and biochemical characteristics such as yellow colonies, were like those of Xanthomonas spp. Further, the pathogen was confirmed using XV1F/XV1R pair of species-specific primers, which amplify the atpD gene sequence solely in Xanthomonas vesicatoria. Out of 30 isolates, 16 isolates were confirmed to be Xcv. In these agro-ecological zones, this was the first report on the presence of Xcv. Furthermore, humidity and temperature were found to impact the incidence and severity of bacterial spot substantially. Most isolates grew at high temperatures and low humidity; however, some prefered high-temperature and low-humidity environments. In conclusion, bacterial spot disease caused by Xcv has now spread throughout all tomato-producing areas of KP province. To avert future plant epidemics, ongoing climate and pest monitoring is required to build an effective disease warning system for producers and other agriculture-related agencies.

Combining DNA Mini-Barcoding and Species-Specific Primers PCR Technology for Identification of Heosemys grandis

Heosemys grandis, a species of Asian water turtle, that has a wide range of applications in the food and pharmaceutical industries. Since some processed products cannot be identified exclusively by morphological and microscopic identification, a reliable and quick approach to guarantee authenticity is critical. Thus, we fostered an effective and stable molecular identification system to identify Heosemys grandis based on DNA mini-barcoding and species-specific primers PCR technology. A total of 48 turtle samples from 16 different species were collected. To distinguish Heosemys grandis from its counterfeits, DNA mini-barcoding and a pair of species-specific primers were designed and verified by PCR after analyzing the COI sequences of samples. The results showed that only Heosemys grandis samples could generate a single clear band following amplification using species-specific primers. Employing DNA mini-barcoding to amplify samples can verify authenticity by sequence alignment. These findings indicated that species-specific primers PCR technology combined with DNA mini-barcoding could accurately detect the authenticity of Heosemys grandis. This technology broadens the application of molecular biology techniques in the food and pharmaceutical industries. It provides a reliable and convenient method for identifying raw materials to standardize the market and protect customers’ rights and interests.

Shoot rot of Zizania latifolia and the first record of its pathogen Pantoea ananatis in China

The aquatic grass Zizania latifolia grows symbiotically with the fungus Ustilago esculenta producing swollen structures called Jiaobai, widely cultivated in China. A new disease of Z. latifolia was found in Zhejiang Province, China. Initial lesions appeared on the leaf sheaths or sometimes on the leaves near the leaf sheaths. The lesions extended along the axis of the leaf shoots and formed long brown to dark brown streaks from the leaf sheath to the leaf, causing sheath rot and death of entire leaves on young plants. The pathogen was isolated and identified as the bacterium Pantoea ananatis, based on 16S ribosomal RNA (rRNA) gene sequencing, multilocus sequence analysis (atpD (β-subunit of ATP synthase F1), gyrB (DNA gyrase subunit B), infB (translation initiation factor 2), and rpoB (β‍-subunit of RNA polymerase) genes), and pathogenicity tests. Ultrastructural observations using scanning electron microscopy revealed that the bacterial cells colonized the vascular tissues in leaf sheaths, forming biofilms on the inner surface of vessel walls, and extended between vessel elements via the perforated plates. To achieve efficient detection and diagnosis of P. ananatis, species-specific primer pairs were designed and validated by testing closely related and unrelated species and diseased tissues of Z. latifolia. This is the first report of bacterial sheath rot disease of Z. latifolia caused by P. ananatis in China.

A conventional multiplex PCR for the detection of four common soil-transmitted nematodes in human feces: development and validation.

Soil-transmitted helminth (STH) infections, mainly caused by Ascaris lumbricoides, Trichuris trichiura, and hookworms, are among the most common intestinal parasites that infect humans. The infections are widely distributed throughout tropical and subtropical countries, including Malaysia, particularly in underprivileged communities. Microscopic and culture techniques have been used as a gold standard for diagnostic techniques. However, these methods yield low sensitivity and specificity, laborious and time-consuming. Therefore, simple, rapid, and accurate alternative methods are needed for the simultaneous detection of STH infections. Although advanced technologies such as real-time multiplex PCR have been established, the use of this technique as a routine diagnostic is limited due to the high cost of the instrument. Therefore, a single-round multiplex conventional PCR assay for rapid detection of four STH species in the fecal sample was developed in this study. To perform the single-round multiplex PCR, each pair of species-specific primers was selected from target genes, including Ancylostoma duodenale (Internal Transcribed Spacer 2; accession No. AJ001594; 156 base pair), Necator americanus (ITS 2; accession No. AJ001599; 225 base pair), Ascaris lumbricoides (Internal Transcribed Spacer 1; accession No. AJ000895; 334 base pair) and Trichuris triciura (partial ITS 1, 5.8s rRNA and partial ITS 2; accession No. AM992981; 518 base pair). The results showed that the newly designed primers could detect the DNA of STH at low concentrations (0.001 ng/ μl) with no cross-amplification with other species. This assay enables the differentiation of single infections as well as mixed infections. It could be used as an alternative and is a convenient method for the detection of STHs, especially for the differentiation of N. americanus and A. duodenale.

Infection and Egg Development of the Root-Knot Nematode Meloidogyne javanica on Oleander Nerium oleander

Adel, R.M. and F.K. Aljuboori. 2022. Infection and Egg Development of the Root-Knot Nematode Meloidogyne javanica on Oleander Nerium oleander. Arab Journal of Plant Protection, 40(4): 293-298. https://doi.org/10.22268/AJPP40.4.293298 The identification and first record of Nerium oleander infection with root-knot nematode M. javanica was confirmed by the PCR test using the pair of species-specific primers (Fjav and Rjav). Symptoms of the Oleander root-knot disease are similar to those caused by these nematodes on other flowering plants. The results of the study described the stages of embryo development inside the egg and until hatching under laboratory conditions. The difference in egg shape index ranged from 35.8 to 87.9, whereas the dominant egg shape index was between 44.5 and 48.8. Keywords: Nerium oleander, eggs, embryo development, root knot nematode, Meloidogyne javanica

Multiplex PCR development for the simultaneous and rapid detection of two pathogenic flukes, Dactylogyrus spp. and Centrocestus formosanus, in ornamental fishes

Both monogenean Dactylogyrus spp. and digenean Centrocestus formosanus are major pathogens for aquaculture and the fish industry, especially in goldfish (Carassius auratus). The resulting infections in the fish cause extensive morbidity and mortality. However, the accurate detection of both parasitic diseases requires highly experienced personnel and takes a long time. Reliable and sensitive diagnostic methods are needed for detecting the infections in various fish species. To overcome this problem, this study developed the multiplex polymerase chain reaction (m-PCR) assay to rapidly identify and diagnose Dactylogyrus spp. and C. formosanus simultaneously on the target genes (18S and cytb gene) in field-collected samples under one reaction. Two pairs of species-specific primers were manually designed. The optimal reaction conditions, specificity, and sensitivity of the m-PCR assay were investigated. The results demonstrated that the multiplex PCR assay can be used to detect Dactylogyrus spp. and C. formosanus specifically, without cross-amplification with other parasite-related species and their host. The sensitivity showed that the m-PCR system could amplify the two target genes with the lowest DNA concentration at 0.078 ng/μL for Dactylogyrus spp. and 0.625 ng/μL for C. formosanus. The detection limit in the case of genomic combination between samples with goldfish was detected at the ratios of 1:13 and 1:2 ng/μL for Dactylogyrus spp. and C. formosanus, respectively. Moreover, this assay can also be developed and applied to detect the adult, metacercariae, cercariae, and egg stages of C. formosanus as well. Additionally, a total of 50 goldfish samples collected from several farms were found to be positive for Dactylogyrus spp., C. formosanus, and co-infection (fifteen, fourteen, and eighteen, respectively). The developed multiplex PCR shows advantages in specificity and sensitivity which prove to be an invaluable tool for diagnoses and monitoring the epidemic situation.

Serological and molecular investigations of Brucellosis in dairy cows at certain areas of Al-Sulaymaniyah governorate- Iraq

This study aimed to detect Brucella antibodies in the sera of dairy cows and to identify Brucella species in the milk of seropositive cows. A total of 100 sera and 100 milk samples were collected from two 50-cows groups (group 1 with and group 2 without a history of reproductive problems and/or decreased milk production). Rose Bengal plate test and indirect ELISA were used to explore Brucella antibodies in the serum samples and thereafter milk samples of seropositive cows were undergone PCR analysis using Brucella genus specific primers and 3 pairs of species specific primers for identification of B. abortus, B. melitensis and B. suis. The RBPT showed 22 cows were carriers for the Brucella antibodies, 18 in group 1 and 4 in group 2 whereas the iELISA showed only 10 cows out of these 22 cows were positive, 9 in group 1 and only 1 cow in group 2. The PCR assay, which was performed on milk samples of the RBPT positive cows, revealed 18 samples were positive for the Brucella genus and the Brucella abortus species and were negative for Brucella melitensis and Brucella suis species. As a conclusion, the results of this study showed that brucellosis has been encountered in cows with or without a history of reproductive problems, and the RBPT followed by PCR assay for milk samples of the seropositive cows could provide more specific detection than performing either test alone and could be more useful for rapid screening of brucellosis in dairy cows.

Development of a One-Step Multiplex PCR Assay for Differential Detection of Four species (Enterobacter cloacae, Enterobacter hormaechei, Enterobacter roggenkampii, and Enterobacter kobei) Belonging to Enterobacter cloacae Complex With Clinical Significance.

Enterobacter cloacae complex (ECC) is composed of multiple species and the taxonomic status is consecutively updated. In last decades ECC is frequently associated with multidrug resistance and become an important nosocomial pathogen. Currently, rapid and accurate identification of ECC to the species level remains a technical challenge, thus impedes our understanding of the population at the species level. Here, we aimed to develop a simple, reliable, and economical method to distinguish four epidemiologically prevalent species of ECC with clinical significance, i.e., E. cloacae, E. hormaechei, E. roggenkampii, and E. kobei. A total of 977 ECC genomes were retrieved from the GenBank, and unique gene for each species was obtained by core-genome comparisons. Four pairs of species-specific primers were designed based on the unique genes. A total of 231 ECC clinical strains were typed both by hsp60 typing and by species-specific PCRs. The specificity and sensitivity of the four species-specific PCRs ranged between 96.56% and 100% and between 76.47% and 100%, respectively. The PCR for E. cloacae showed the highest specificity and sensitivity. A one-step multiplex PCR was subsequently established by combining the species-specific primers. Additional 53 hsp60-typed ECC and 20 non-ECC isolates belonging to six species obtained from samples of patients, sewage water and feces of feeding animals were tested by the multiplex PCR. The identification results of both techniques were concordant. The multiplex PCR established in this study provides an accurate, expeditious, and cost-effective way for routine diagnosis and molecular surveillance of ECC strains at species level.

Identification of Penicillium verrucosum, Penicillium commune, and Penicillium crustosum Isolated from Chicken Eggs

Penicillium species belong to main causative agents of food spoilage leading to significant economic losses and potential health risk for consumers. These fungi have been isolated from various food matrices, including table eggs. In this study, both conventional Polymerase Chain Reaction (PCR) and Polymerase Chain Reaction-Internal Transcribed Spacer-Restriction Fragment Length Polymorphism (PCR-ITS-RFLP) methods were used for species identification of Penicillium (P.) spp. isolated from the eggshells of moldy chicken eggs. Seven restriction endonucleases (Bsp1286I, XmaI, HaeIII, HinfI, MseI, SfcI, Hpy188I) were applied to create ribosomal restriction patterns of amplified ITS regions. To identify P. verrucosum, P. commune, and P. crustosum with the help of conventional PCR assay, species-specific primer pairs VERF/VERR, COMF/COMR, and CRUF/CRUR were designed on the base of 5.8 subunit-Internal Transcribed Spacer (5.8S-ITS) region. Altogether, 121 strains of microscopic filamentous fungi were isolated by traditional culture mycological examination. After morphological evaluation of both macroscopic and microscopic features, 96 strains were classified in Penicillium spp. Two molecular methods used have confirmed eight isolates as P. verrucosum, 42 isolates as P. commune, and 19 isolates as P. crustosum. Both PCR-ITS-RFLP and conventional PCR assays appear to be suitable alternatives for rapid identification of the above mentioned Penicillium species.

A Rapid Method for the Identification of Fresh and Processed Pagellus erythrinus Species against Frauds.

The commercialization of porgies or seabreams of the family Sparidae has greatly increased in the last decade, and some valuable species have become subject to seafood substitution. DNA regions currently used for fish species identification in fresh and processed products belong to the mitochondrial (mt) genes cytochrome b (Cytb), cytochrome c oxidase I (COI), 16S and 12S. However, these markers amplify for fragments with lower divergence within and between some species, failing to provide informative barcodes. We adopted comparative mitogenomics, through the analysis of complete mtDNA sequences, as a compatible approach toward studying new barcoding markers. The intent is to develop a specific and rapid assay for the identification of the common pandora Pagellus erythrinus, a sparid species frequently subject to fraudulent replacement. The genetic diversity analysis (Hamming distance, p-genetic distance, gene-by-gene sequence variability) between 16 sparid mtDNA genomes highlighted the discriminating potential of a 291 bp NAD2 gene fragment. A pair of species-specific primers were successfully designed and tested by end-point and real-time PCR, achieving amplification only in P. erythrinus among several fish species. The use of the NAD2 barcoding marker provides a rapid presence/absence method for the identification of P. erythrinus.

QPCR-based detection of Colletotrichum truncatum in soybean seeds

Soybean seed infected with Colletotrichum truncatum is an important source of primary inoculum for anthracnose epidemics. Based on differences in the GAPDH gene sequences of Colletotrichum species, one pair of species-specific primers, CtruncF1/CtruncR1, was designed to accurately detect C. truncatum in soybean seed samples. The primers amplified only a single PCR band of 211 bp from C. truncatum. SYBR Green qPCR using these primers enabled the detection of DNA of the target fungus in inoculated soybean seeds and in naturally infested seeds. The sensitivity of the method was 0.000253 ng/μL of C. truncatum DNA template, with an efficiency of 1.78 and a Ct of 30.09. These species-specific primers may be useful for certification of soybean seeds aimed at avoiding introduction of the pathogen into soybean-producing regions.

Molecular detection and phylogenetic analyses of Wolbachia in natural populations of nine galling Aphid species

Wolbachia is one of the most abundant facultative intracellular symbionts in arthropods. It alters host biology in diverse ways, including the induction of reproductive manipulation, association of nutrient supplier and protection against pathogens. Aphids are a group of insects which exhibit interesting biological characteristics such as complex life cycles, alteration of sexual and asexual reproduction and shifts between two different hosts. Wolbachia is widely present in many orders of insects, but so far limited studies on Wolbachia in aphids have been carried out. Galling aphids are a group of aphids that induce galls on their primary host plants at specific life stage. In this study, 15 natural populations representing nine galling aphid species were analyzed for the presence of Wolbachia using species-specific primer pairs. Wolbachia presence in galling aphids was quite low and varied significantly among aphid populations. Only three of the 15 populations we analyzed had detectable Wolbachia and the overall infection rate was 20%. Two Wolbachia strains, O and B, were identified from the galling aphids Kaburagia rhusicola and Schlechtendalia chinensis. Strain O was for the first time to be found in aphids, and it is likely involved with the life stages of galling aphids living in closed microenvironments with specific survival strategies that are different from free-living aphids.

Identification, genetic characterization and virulence of Serbian Erwinia amylovora isolates

Fire blight disease, caused by plant pathogenic bacteria Erwinia amylovora represents a global threat for pome fruit industry, affecting yield and causing serious economic damage. During six year period (2011–2016), a total of 45 isolates typical for E. amylovora were isolated from four different host plants (apple, pear, quince and medlar) in 21 localities within four Serbian regions (Southeast, Central, West, and North). Isolates were primarily identified using biochemical and serological tests, polymerase chain reaction (PCR) with species-specific primer pair (A/B) and their pathogenicity. Polyclonal antibodies used in DAS ELISA and IF were found to be specific and sensitive enough for rapid E. amylovora detection. In virulence test performed on immature pear fruitlets, higher variability (CV) was found between regions than among hosts from which isolates originated. Slighter virulence of isolates originated from North region of Serbia (Vojvodina) was found in comparison to the isolates from Central Serbia, which were the most variable. Principal components analysis (PCA) segregated quince as a host among the most prevalent isolates in group of highly virulent, but failed to identify difference related to geographic origin of Serbian isolates. The same results enabled multivariate statistical analysis. Different DNA fingerprinting techniques such as repetitive element sequence based PCR (rep-) with BOX, ERIC, REP, GTG5 and SERE primers, then Random Amplified Polymorphic DNA (RAPD) with M13-PCR primer revealed genetic homogeneity among all Serbian E. amylovora isolates. ERIC, SERE and M13 primers generated the most complex and visually perceptible patters. Restriction fragment length polymorphism analysis (RFLP) of gyrA and recA genes, digested with three restriction endonucleases BstUI, BgIII and BsmI and rpoS gene digested with BstUI gave the unuque, characteristic patterns for all Serbian isolates.