Designed Primer Sets Research Articles

A rapid LAMP‐based colorimetric assay with quick DNA extraction for on‐site identification of Drosophila suzukii Matsumura

AbstractThe spotted wing drosophila, Drosophila suzukii Matsumura (Diptera: Drosophilidae), distributes in most soft‐skinned fruit areas in China, is an economically important pest of fresh cherries during Sino‐European trade and is considered a quarantine pest in A2 list by European and Mediterranean Plant Protection Organization. Loop‐mediated isothermal amplification (LAMP) is an accurate, quick and convenient molecular identification method, applied to distinguish D. suzukii from other fruit flies. This method can be used for inspection in the field, and at the points of entry (POEs), because its results can be detected with the naked eye due to colour changes. In our study, first, we reported a simple and fast LAMP colorimetric detection method for molecular identification of D. suzukii. We designed primer sets for D. suzukii based on mitochondrial cytochrome oxidase I (COI) sequences. The specificity and sensitivity of the primers were tested by using target and non‐target fruit flies in the family Drosophilidae and Tephritidae, usually intercepted during the Sino‐European cherry trade. Second, for detection in the field and at POEs, the adoption of a quick DNA extraction method could save the total time of LAMP identification to about 90 min. Taken together, this accurate, quick and convenient LAMP‐based colorimetric identification assay combined with a quick DNA extraction method could visually detect clearly with just one portable heating device, which will be useful for rapid on‐site identification and inspection for D. suzukii by the quarantine department.

Designing specific bacterial 16S primers to sequence and quantitate plant endo-bacteriome.

Plant endophytic bacteria colonize the internal tissues of plants and interact with plants closely. The past two decades have witnessed the increasing application of next-generation 16S rRNA gene sequencing in the investigation of bacterial communities. However, deciphering plant endo-bacterial communities by this method is difficult because of the co-amplification of massive plant organellar DNAs with bacterial 16S. Here, we designed polymerase chain reaction (PCR) primer sets, including 799F/1107R, 322F/796R, and 322F-Dr/796Rs (primer pair 322F/796R with a penultimate-base substitution in 322F), that can specifically amplify bacterial 16S from plant total DNAs. We computationally and experimentally evaluated the specificity, coverage, and accuracy of the newly designed primer sets. Both 799F/1107R and 322F-Dr/796Rs produced plant DNA-free 16S amplicon libraries or reduced plant DNA contamination to lower than 5% for the plant materials with extremely-low-abundance bacterial communities. The primer set 322F-A/796R was used through absolute quantitative PCR to quantitate the population size of rice leaf or root endo-bacteriome, which revealed 106-107 and 109-1010 bacteria per gram fresh weight, respectively. These 16S primer sets and amplification methods enable the simple and inexpensive next-generation sequencing and quantification of plant endo-bacteriome, which will significantly advance studies on the plant-related microbiome.

New Histoplasma Diagnostic Assays Designed via Whole Genome Comparisons

Histoplasmosis is a systemic fungal disease caused by the pathogen Histoplasma spp. that results in significant morbidity and mortality in persons with HIV/AIDS and can also affect immunocompetent individuals. Although some PCR and antigen-detection assays have been developed, conventional diagnosis has largely relied on culture, which can take weeks. Our aim was to provide a proof of principle for rationally designing and standardizing PCR assays based on Histoplasma-specific genomic sequences. Via automated comparisons of aligned genome contigs/scaffolds and gene (sub)sequences, we identified protein-coding genes that are present in existing sequences of Histoplasma strains but not in other genera. Two of the genes, PPK and CFP4, were used for designing primer sets for conventional and real-time PCR assays. Both resulted in a 100% analytical specificity in vitro and detected 62/62 H. capsulatum isolates using purified DNA. We also obtained positive detections of 2/2 confirmed H. capsulatum clinical FFPE (formalin-fixed paraffin-embedded) samples using both primer sets. Positive control plasmid 10-fold serial dilutions confirmed the analytical sensitivity of the assays. The findings suggest that these novel primer sets should allow for detection sensitivity and reduce false positive results/cross-reactions. New assays for detecting pathogenic fungi, constructed along these lines, could be simple and affordable to implement.

Development of a Potential Penside Colorimetric LAMP Assay Using Neutral Red for Detection of African Swine Fever Virus.

African swine fever (ASF) has caused huge economic losses to the swine industry worldwide. Since there is no commercial ASF vaccine available, an early diagnosis is extremely important to prevent and control the disease. In this study, ASF virus (ASFV) capsid protein-encoding gene (p72) was selected and used to design primers for establishing a one-step visual loop-mediated isothermal amplification (LAMP) assay with neutral red, a pH-sensitive dye, as the color shift indicator. Neutral red exhibited a sharp contrast of color change from faint orange (negative) to pink (positive) during LAMP for detection of ASFV. The designed primer set targeting highly conserved region of the p72 gene was highly specific to ASFV and showed no cross-reactivity with other swine viruses. The detection limit for the one-step visual LAMP developed was 10 copies/reaction based on the recombinant plasmid containing the p72 gene of ASFV. More importantly, the developed one-step visual LAMP showed high consistency with the results of the real-time polymerase chain reaction (qPCR) method recommended by World Organization for Animal Health (OIE). Furthermore, the results demonstrate that the colorimetric detection with this LAMP assay could be directly applied for the whole blood and serum samples without requiring genome extraction. Based on our results, the developed one-step visual LAMP assay is a promising penside diagnostic tool for development of early and cost-effective ASF monitoring program that would greatly contribute to the prevention and control of ASF.

Fine Tuning of Real Time PCR as a First Tool for the Detection of G143A Substitution in <i>Venturia inaequalis</i> Samples

Apple scab caused by Venturia inaequalis (Cke.) Wint. is the most important disease of apple trees worldwide and requires a high number of fungicide applications. The G143A substitution in the inhibitor binding site of cytochrome b of V. inaequalis confers a high level of resistance to strobilurins targeting the bc1 complex. The aim of this work was to substitute the labor intensive in vitro assays, with the faster quantitative PCR. An allele-specific qPCR method with a newly designed primer set was successfully developed to quantitatively determine the frequency of QoI-resistant A143 allele in populations of V. inaequalis. To be able to suggest that the molecular method could be applied as unique and robust technique, we carried out in vitro sensitivity test to trifloxystrobin; first testing the relative germination and subsequently confirmed with the quantification of mutated allele frequencies by qPCR on forty-nine Italian V. inaequalis populations. qPCR gave a similar pattern to that obtained using in vitro conidial germination test in predominantly sensitive and resistant populations, the variability between these two tests was observed in some heterogeneous populations. The qPCR assay developed in this study efficiently quantifies the A143 allele and we can conclude that this method could be useful for the study of the fungicide resistance at population level in the fields, giving a quick response also with a large amount of samples.

Evaluation of different target genes for the detection of Salmonella sp. by loop‐mediated isothermal amplification

The loop-mediated isothermal amplification (LAMP) technique was used to investigate six salmonella-specific sequences for their suitability to serve as targets for the pathogen identification. Sequences selected for designing LAMP primers were genes invA, bcfD, phoP, siiA, gene62181533 and a region within the ttrRSBCA locus. Primers including single nucleotide polymorphisms were configured as degenerate primers. Specificity of the designed primer sets was determined by means of 46 salmonella and 32 other food- and waterborne bacterial reference species and strains. Primers targeting the ttrRSBCA locus showed 100 % inclusivity of target and exclusivity of other test species and strains. Other primer sets revealed deficiencies, especially regarding Salmonella enterica subsp. II-IV and Salmonella bongori. Additionally, primers targeting the siiA gene failed to detect S. enterica subsp. enterica serotypes Newport and Stanley, whereas bcfD primers did not amplify DNA of S. enterica subsp. enterica serotype Schleissheim. TtrRSBCA primers, providing short detection times and constant melting temperatures of amplification products, achieved best overall performance.

Novel Primer Sets for Rapid Detection of Zymoseptoria tritici in Wheat.

Zymoseptoria tritici is a fungal pathogen causing losses in wheat yields. Here, we present new primer sets for species-specific identification of this microorganism in wheat leaf samples using conventional PCR. Primer sets were validated in silico using tools available in genetic databases. Furthermore, in vitro tests were also carried out on 190 common wheat samples with visual symptoms of Septoria tritici blotch (STB) collected in Poland in three growing seasons (2015, 2016, 2017). The designed primer sets showed full hybridization to the available genetic resources deposited in the NCBI GenBank database, and their high specificity and sensitivity were demonstrated on wheat leaf samples and selected fungal strains.

Use of Newly Designed Primers for Quantification of Complete Ammonia-Oxidizing (Comammox) Bacterial Clades and Strict Nitrite Oxidizers in the Genus Nitrospira.

Complete ammonia-oxidizing (comammox) bacteria play key roles in environmental nitrogen cycling and all belong to the genus Nitrospira, which was originally believed to include only strict nitrite-oxidizing bacteria (sNOB). Thus, differential estimation of sNOB abundance from that of comammox Nitrospira has become problematic, since both contain nitrite oxidoreductase genes that serve as common targets for sNOB detection. Herein, we developed novel comammox Nitrospira clade A- and B-specific primer sets targeting the α-subunit of the ammonia monooxygenase gene (amoA) and a sNOB-specific primer set targeting the cyanase gene (cynS) for quantitative PCR (qPCR). The high coverage and specificity of these primers were checked by use of metagenome and metatranscriptome data sets. Efficient and specific amplification with these primers was demonstrated using various environmental samples. Using the newly designed primers, we successfully estimated the abundances of comammox Nitrospira and sNOB in samples from two chloramination-treated drinking water systems and found that, in most samples, comammox Nitrospira clade A was the dominant type of Nitrospira and also served as the primary ammonia oxidizer. Compared with other ammonia oxidizers, comammox Nitrospira had a higher abundance in process water samples in these two drinking water systems. We also demonstrated that sNOB can be readily misrepresented by an earlier method, calculated by subtracting the comammox Nitrospira abundance from the total Nitrospira abundance, especially when the comammox Nitrospira proportion is relatively high. The new primer sets were successfully applied to comammox Nitrospira and sNOB quantification, which may prove useful in understanding the roles of Nitrospira in nitrification in various ecosystems.IMPORTANCENitrospira is a dominant nitrite-oxidizing bacterium in many artificial and natural environments. The discovery of complete ammonia oxidizers in the genus Nitrospira prevents the use of previously identified primers targeting the Nitrospira 16S rRNA gene or nitrite oxidoreductase (nxr) gene for differential determination of strict nitrite-oxidizing bacteria (sNOB) in the genus Nitrospira and among comammox bacteria in this genus. We designed three novel primer sets that enabled quantification of comammox Nitrospira clades A and B and sNOB with high coverage, specificity, and accuracy in various environments. With the designed primer sets, sNOB and comammox Nitrospira were differentially estimated in drinking water systems, and we found that comammox clade A predominated over sNOB and other ammonia oxidizers in process water samples. Accurate quantification of comammox Nitrospira and sNOB by use of the newly designed primers will provide essential information for evaluating the contribution of Nitrospira to nitrification in various ecosystems.

Lost in translation: the quest for Nitrosomonas cluster 7-specific amoA primers and TaqMan probes.

The choice of primer and TaqMan probes to quantify ammonia-oxidizing bacteria (AOB) in environmental samples is of crucial importance. The re-evaluation of primer pairs based on current genomic sequences used for quantification of the amoA gene revealed (1) significant misrepresentations of the AOB population in environmental samples, (2) and a lack of perfect match primer pairs for Nitrosomonas europaea and Nitrosomonas eutropha. We designed two new amoA cluster 7-specific primer pairs and TaqMan probes to quantify N. europaea (nerF/nerR/nerTaq) and N. eutropha (netF/netR/netTaq). Specificity and quantification biases of the newly designed primer sets were compared with the most popular primer pair (amoA1f/amoA2r) using DNA from various AOB cultures as individual templates as well as DNA mixtures and environmental samples. Based on the qPCR results, we found that the newly designed primer pairs and the most popular one performed similarly for individual templates but differed for the DNA mixtures and environmental samples. Using the popular primer pair introduced a high underestimation of AOB in environmental samples, especially for N. eutropha. Thus, there is a strong need for more specific primers and probes to understand the occurrence and competition between N. europaea and N. eutropha in different environments.

Development and Validation of a Real-Time PCR Based Assay to Detect Adulteration with Corn in Commercial Turmeric Powder Products.

Turmeric, or Curcuma longa, is commonly consumed in the South East Asian countries as a medical product and as food due to its therapeutic properties. However, with increasing demand for turmeric powder, adulterated turmeric powders mixed with other cheap starch powders, such as from corn or cassava, are being distributed by food suppliers for economic benefit. Here, we developed molecular markers using quantitative real-time PCR to identify adulteration in commercial turmeric powder products. Chloroplast genes, such as matK, atpF, and ycf2, were used to design species-specific primers for C. longa and Zea mays. Of the six primer pairs designed and tested, the correlation coefficients (R2) were higher than 0.99 and slopes were −3.136 to −3.498. The efficiency of the primers was between 93.14 and 108.4%. The specificity of the primers was confirmed with ten other species, which could be intentionally added to C. longa powders or used as ingredients in complex turmeric foods. In total, 20 blind samples and 10 commercial C. longa food products were tested with the designed primer sets to demonstrate the effectiveness of this approach to detect the addition of Z. mays products in turmeric powders. Taken together, the real-time PCR assay developed here has the potential to contribute to food safety and the protection of consumer’s rights.

Development of a SYBR Green quantitative PCR assay for detection of Lates calcarifer herpesvirus (LCHV) in farmed barramundi

Lates calcarifer herpes virus (LCHV) is a novel virus of farmed barramundi in Southeast Asia. However, a rapid detection method is yet to be available for LCHV. This study, therefore, aimed to develop a rapid quantitative PCR (qPCR) detection method for LCHV and made it timely available to public for disease diagnostics and surveillance in barramundi farming countries. A newly designed primer set targeting a 93-bp fragment of the LCHV putative major envelope protein encoding gene (MEP) was used for developing and optimizing a SYBR Green based qPCR assay. The established protocol could detect as low as 10 viral copies per μl of DNA template in a reaction containing spiked host DNA. No cross-amplification with genomic DNA extracted from host as well as common aquatic pathogens (12 bacteria and 4 viruses) were observed. Validation test of the method with clinical samples revealed that the virus was detected in multiple organs of the clinically sick fish but not in the healthy fish. We thus recommend that barramundi farming countries should promptly initiate active surveillance for LCHV in order to understand their circulation for preventing possibly negative impact to the industry.

PCR detection of Pseudomonas syringae pv. syringae, the causal agent of bacterial black node in barley and wheat, using newly designed primer sets

We designed two novel primer sets based on the sequence of the hrpZ gene to detect Pseudomonas syringae pv. syringae, the causal agent of bacterial black node in barley and wheat. A conventional PCR using these primers amplified only P. syringae group III strains. The result of a nested PCR with the novel primers showed that P. syringae pv. syringae was detectible even when only one culturable cell was used as the PCR template. This highly sensitive PCR detected P. syringae in diseased parts of wheat and barley plants and in asymptomatic plants.

Molecular identification of dried squid products sold in China using DNA barcoding and SYBR green real time PCR

ABSTRACT Dried squid products are popular in China as a snack food, side dishes, or refreshments, and the market appeal can be reflected by the high price that occasionally reaches 497 RMB per kg. However, the absence of harmonisation around the definition of squid, as well as the problems with visual inspection for processed seafood products, make alternative species substitution for dried squid products a frequent occurrence. The aim of the present study was to apply a DNA barcoding approach for species identification of 48 dried squid products collected from the largest online shopping platform in China. Moreover, we also developed a novel SYBR green real-time PCR assay (simplex and duplex followed by a melting curve analysis) specific for Illex argentinus and Todarodes pacificus based on cytochrome C oxidase subunit I (COI) gene. Results highlighted the successful DNA extraction and PCR amplification of a 655 bp COI gene fragment from all products. A maximum similarity value in the range of 98-100% was obtained for all readable sequences using the BOLD and BLAST public databases and four species (Dosidicus gigas, Uroteuthis edulis, I. argentinus, and T. pacificus) were identified. The specificity of the designed primer sets was confirmed against 23 non-target species, and the newly developed methods were successfully applied to screen I. argentinus and T. pacificus in dried squid products. Overall, DNA barcoding is a robust tool for seafood species identification and the novel method is effective in screening I. argentinus and T. pacificus in food products.

Microsatellite DNA markers applicable to paternity inference in the androdioecious gooseneck barnacle Octolasmis warwickii (Lepadiformes: Poecilasmatidae).

The gooseneck barnacle Octolasmis warwickii has a rare sexual system called androdioecy, in which hermaphrodites and dwarf males co-occur. It has been hypothesized that dwarf males can coexist with conspecific hermaphrodites when dwarf males are capable of leaving more offspring than hermaphrodites via male reproduction. This hypothesis of reproductive superiority of dwarf males can be validated by comparing the reproductive success between dwarf males and hermaphrodites through DNA marker-based parentage testing. In the present study, we developed microsatellite DNA markers for O. warwickii, and evaluated the power of these markers to infer parentage based on simulation analysis. Using next generation sequencing, we obtained 344 microsatellite sequences suitable for designing primer sets for amplification in polymerase chain reaction (PCR). Of these, we examined the PCR amplification efficiency of 54 primer sets, of which 11 passed our primer screening in a population sample (n = 35). The developed markers exhibited moderate to high levels of polymorphisms, and met Hardy-Weinberg equilibrium with little evidence of significant allelic association to each other. Our simulated paternity inference suggested that the combinational use of the markers allows a high resolution of parentage (success rate of > 99.9%) if all candidate fathers are available.

Identification of opportunistic and nonopportunistic Exophiala species using high resolution melting analysis.

Exophiala is a genus comprising several species of opportunistic black yeasts. Exophiala species identification by morphological, physiological, and biochemical characteristics is challenging because of the low degree of phenotypic differences between species and its polyphyletic nature. We aimed to develop a high-resolution melting (HRM) assay based on the internal transcribed spacer (ITS) region to differentiate between pairs of clinical and environmental Exophiala species. HRM primers were designed based on the conserved ITS region of five Exophiala species (E. dermatitidis, E. phaeomuriformis, E. heteromorpha, E. xenobiotica, and E. crusticola). Environmental and clinical Exophiala isolates representing these five species (n=109) were analyzed. The HRM assay was optimized using clinical and environmental reference isolates (n=22), and then the results were compared with those obtained with nonreference isolates of Exophiala (n=87) using two designed primer sets. The designed HRM assay was based on the normalized melting peak approach and two primer sets, and successfully distinguished between the five Exophiala species. The HRM1 primer set provided sufficient resolution, with a melting temperature (Tm) difference of approximately 2.5°C among the analyzed species and of approximately 1°C between E. dermatitidis and E. phaeomuriformis. HRM typing results were in agreement with those of ITS-sequence typing (100% sensitivity and specificity). The developed HRM assay can be used to ascertain the identity of Exophiala species, which may differ in clinical significance, with high accuracy. Its application to identify species directly in clinical samples and/or environmental niches may be possible in the future.

Distribution of mating type alleles in Iranian populations of Pyrenophora graminea, the causal agent of barley leaf stripe disease, using a multiplex PCR approach

Pyrenophora graminea is the main fungal species associated with barley leaf stripe disease worldwide. Even though a heterothallic mating strategy has been proven for P. graminea, this species is mainly known based on the asexual morph in nature and there is no available information on the prevalence of an active sexual cycle within the populations of this species in Iran as well as many other barley-producing countries. The feasibility of a cryptic sexual cycle within Iranian isolates of P. graminea was assessed through analyzing the distribution and frequency of the mating type alleles on micro-spatial and macro-geographical scales. A total number of 306 P. graminea isolates were obtained from 93 fields in 45 geographical regions across East Azerbaijan province during 2016–2017. A multiplex PCR assay was developed for simultaneous identification of P. graminea and screening of its mating type alleles using previously designed primer sets. Using the multiplex PCR assay, a 435-bp band was consistently amplified from all P. graminea isolates; while a 1300-bp fragment or a 1150-bp fragment was only amplified from the isolates harboring MAT-1 and MAT-2 alleles, respectively. The mating type identity of 164 isolates was determined as MAT-1 and 142 isolates as MAT-2. Results of the present study revealed a nearly equal distribution (1:1 ratio; X2=1.582) of mating type alleles within and between different populations of P. graminea. Results of the micro-spatial and macro-geographical distribution of mating types showed that both mating types were often present on almost all studied scales, including: within the same lesion of each leaf from a single barley plant, from the same field or different fields in the same region, and from different regions. Based on the results of the current study and referring to the earlier reports on the population structure of P. graminea, it is concluded that this pathogen undergoes regular cycles of sexual recombination in most of the examined regions.

Molecular identification of Lutjanus species by PCR-RFLP analysis of mitochondrial 12S rRNA region

A PCR-RFLP was developed by targeting the mitochondrial 12S rRNA region to authenticate the six species viz., Lutjanus fulvus, L. rivulatus, L. quinquelineatus, L. fulviflamma, L. madras and L. decussatus available along the Indian coast. A newly designed primer set, 12SU-F/12SU-R, was used for PCR amplification to obtain a product size of 550 bp, which was then digested using three endonucleases viz., BseDI, Mn1I and MspA1I. All the six species were unambiguously differentiated by BseDI enzyme. Mn1I differentiated only four species viz., L. fulvus, L. quinquelineatus, L. fulviflamma and L. madras; while MspAI differentiated four species viz., L. rivulatus, L. quinquelineatus, L. fulviflamma and L. madras from the rest. The developed PCR-RFLP protocol using BseDI enzyme can therefore be easily adopted by regulatory authorities to the prevention of possible adulteration of species for red snappers.

Development of the Automated Primer Design Workflow Uniqprimer and Diagnostic Primers for the Broad-Host-Range Plant Pathogen Dickeya dianthicola.

Uniqprimer, a software pipeline developed in Python, was deployed as a user-friendly internet tool in Rice Galaxy for comparative genome analyses to design primer sets for PCRassays capable of detecting target bacterial taxa. The pipeline was trialed with Dickeya dianthicola, a destructive broad-host-range bacterial pathogen found in most potato-growing regions. Dickeya is a highly variable genus, and some primers available to detect this genus and species exhibit common diagnostic failures. Upon uploading a selection of target and nontarget genomes, six primer sets were rapidly identified with Uniqprimer, of which two were specific and sensitive when tested with D. dianthicola. The remaining four amplified a minority of the nontarget strains tested. The two promising candidate primer sets were trialed with DNA isolated from 116 field samples from across the United States that were previously submitted for testing. D. dianthicola was detected in 41 samples, demonstrating the applicability of our detection primers and suggesting widespread occurrence of D. dianthicola in North America.

High throughput quantification of the functional genes associated with RDX biodegradation using the SmartChip real-time PCR system.

The explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a contaminant at many military sites. RDX bioremediation as a clean-up approach has been gaining popularity because of cost benefits compared to other methods. RDX biodegradation has primarily been linked to six functional genes (diaA, nfsI, pnrB, xenA, xenB, xplA). However, current methods for gene quantification have the risk of false negative results because of low theoretical primer coverage. To address this, the current study designed new primer sets using the EcoFunPrimer tool based on sequences collected by the Functional Gene Pipeline and Repository and these were verified based on residues and motifs. The primers were also designed to be compatible with the SmartChip Real-Time PCR system, a massively parallel singleplex PCR platform (high throughput qPCR), that enables quantitative gene analysis using 5,184 simultaneous reactions on a single chip with low volumes of reagents. This allows multiple genes and/or multiple primer sets for a single gene to be used with multiple samples. Following primer design, the six genes were quantified in RDX-contaminated groundwater (before and after biostimulation), RDX-contaminated sediment, and uncontaminated samples. The final 49 newly designed primer sets improved upon the theoretical coverage of published primer sets, and this corresponded to more detections in the environmental samples. All genes, except diaA, were detected in the environmental samples, with xenA and xenB being the most predominant. In the sediment samples, nfsI was the only gene detected. The new approach provides a more comprehensive tool for understanding RDX biodegradation potential at contaminated sites.

Detection of Golovinomyces orontii using species-specific primers and high-resolution melting analysis

This study presents the development of species-specific PCR primers for detection of Golovinomyces orontii, the causal agent of powdery mildew disease on different cucurbit hosts. The species-specific primers CgF2 and CgR2 were designed based on partial ITS and 5.8S rDNA sequences using an isolates collection of powdery mildew and downy mildew pathogens from different sources. After optimization of the PCR conditions, a 233 bp fragment was amplified only from DNA of G. orontii. Limit of detection was determined to be 0.01 ng of pure conidial genomic DNA. Further association of PCR and high-resolution melting (HRM) analysis showed that the designed primer set can be used for differentiation of G. orontii from different powdery mildew fungi in infected cucurbits tissue collected in the field.