detection

The genus Malva (family Malvaceae) includes species of herbaceous plants which are often pluriannual and very common in borders and boundaries of cultivated fields, making them proper reservoirs for plant pathogens. They are most commonly used as ornamental plants, although they may be used as a food resource and remedy for various diseases. Common mallow (Malva sylvestris) is the most important Malva species which is used as garden flower as well as widely recognized for food and medicinal purposes.  Malva sylvestris are reported to be affected by several viruses from different genera including malva vein clearing virus (MVCV, Potyvirus), alfalfa mosaic virus (AMV, Alfamovirus), malva-associated soymovirus 1 (MaSV1, Soymovirus) cucumber mosaic virus (CMV, Cucumovirus), and cotton leaf curl Gezira virus (CLCuGeV, Begomovirus). In this study, we attempted to identify important viruses infecting mallow plants and compare mallow isolates with other sequences in the GenBank.

In 2022-2023, twenty samples of mallow plants with viral-like symptoms on the leaves were collected from the urban landscape of Golestan province. Total RNA was extracted by DENAzist Total RNA Isolation Kit from fresh, infected mallow leaves. All samples were analyzed by RT-PCR with specific primer pairs for the detection of CMV, AMV, and MVCV. The amplified fragments in expected size of coat protein (CP) gene of each virus were visualized under UV light in agarose gel after electrophoresis, then amplified fragment of one isolate of each virus was bi-directionally sequenced. Obtained sequences were compared to data available in GenBank. The phylogenetic trees of viruses were constructed based on the nucleotide sequences of the coat protein gene using the neighbor-joining method by MEGA11.

All three viruses were detected in some symptomatic samples collected from Golestan province. The most typical symptoms in positive samples were mosaic, vein clearing, and leaf malformation. Of a total of 20 sampled plants, MVCV, CMV and AMV were detected by RT-PCR in nine, five and two samples, respectively. An amplicon of each virus was selected and sequenced in both directions. BLASTn analysis of the sequenced data confirmed that the PCR-amplified fragments belonged to MVCV, AMV, and CMV. Phylogenetic analysis based on the nucleotide sequence of CP gene of MVCV (n=21) including Iranian and GenBank isolates showed that all isolates are divided into six groups: G1 to G6. All Iranian isolates along with the isolate from the Netherlands were placed in group G5. The phylogenetic tree placed the CMV sequences (n=51) into two distinct phylogroups I and II; the obtained isolate CMV-IR-Ma clustered together with isolates from Iran, Netherlands, South Korea, Japan, China, Hungary, Australia, France and the USA into group II. According to the results of this study, AMV isolates (n=17) can be divided into two groups, I and II. Group I which includes isolates from Canada, China, Italy, Spain, Argentina, Australia and the USA, was divided into six subgroups. The obtained isolate AMV-IR-Ma was clustered in subgroup IB with a Chinese isolate (HZ) and forms a common branch with isolates (Ir-VM, Ir-WS, and Ir-WS2) from Iran. This is the first report of mallow (Malva sylvestris) infection with CMV and AMV in Iran using RT-PCR. In addition, mixed infection with two (MVCV+CMV and MVCV+AMV) or all three viruses (MVCV+CMV+AMV) was also confirmed in some samples. The phylogenetic trees showed that most of the viral isolates were not grouped according to their geographic locations. This suggests the dissemination and spread of these viruses through infected seeds. In Iran, M. sylvestris is a common weed found in fields, waste grounds, roadside verges and gardens. Considering the potential non-persistent aphid transmission as well as mechanical transmission, virus-infected mallows can act as a natural reservoir, thereby posing a threat to other ornamental plants and crops. Since the studied viruses were not detected in the other symptomatic plants, the observed symptoms can be caused by physiological disorders such as nutrient deficiencies, the presence of different viral strains, or other unknown and undetected viral species.

This study provides new knowledge on the diversity and molecular characteristics of viruses in mallow plants (M. sylvestris) affected by the viral disease. The information obtained from this study can be helpful in improving management strategies for disease caused by these viruses in Iran. Albeit M. sylvestris is a host of these viruses, but more comprehensive research on other viral species that may infect this plant need to be conducted. Weed management could be an effective way to eliminate inoculum sources of these viruses.

 In order to minimize the systemic infection in potato seed tubers, annually more than 300 potato seed lots are subjected to laboratory testing for potato viruses in the national potato seed certification system. The accuracy, sensitivity, specificity and speed are of the most important criteria for a diagnostic method to be employed in seed certification laboratory testing procedure. In this study the efficiency of using two methods including single sprout culture and RT-PCR as an integrated diagnostic method was investigated for detecting Potato Virus Y in potato seed samples. According to the present study, RT-PCR performance in Potato Virus Y detection was better than ELISA in terms of sensitivity, accuracy and time saving. RT-PCR successfully detected one infected sample in a combined sample consists of 50 individual samples while ELISA enabled to detect one infected sample in a much smaller combined sample consists of only five subsamples. Moreover because of ELISA retractions to detect low virus concentration and its false negative result, about six percent potato seed lots were incorrectly labeled basic seeds instead of certified seeds. Considering the maximum permissive virus infection percent in national potato seed standards (4% for the lowest seed class) and the maximum required subsamples to be tested (5 subsamples), using RT-PCR method in potato seed certification is economically recommended.

Citrus bacterial canker is an economically important disease in many tropical and subtropical countries and caused by Xanthomonas citri subsp. citri. Several pathotypes of this pathogen have been described that primarily distinguished by their geographical origin, host range and certain genotypic characteristics Leaves, fruits and twinges samples of ‘Mexican 'lime, with bacterial canker symptoms were collected from Kohgiluyeh and Boyer-Ahmad province in 2012. The bacterial strains were isolated from infected tissues using classic methods and primary identification of bacterial isolates was performed based on phenotypic characterization. Pathogenicity of isolates was approved on leaves of Mexican lime. Based on the disease symptoms, phenotypic and pathogenicity tests, PCR test by using specific primers and sequencing data of histidine kinase gene, the isolates were identified as Xanthomonas citri subsp. citri. This is the first report of the disease and the causal bacterium in Kohgiluyeh and Boyer-Ahmad province.

HLB (Huanglongbing ex greening) caused by three species of bacterium Candidatus Liberibacter asiaticus, Ca. L. africanus and Ca. L. americanus, is the most important citrus disease over the globe. The causal agent is transmitted by two psyllids Diaphorina citri and Trioza erytreae and infected bud woods. The causal agent of HLB disease was identified as a phloem-restricted, Gram-negative bacterium belonging to a new genus in the α-Proteobacteria subdivision. In Asia, the pathogen of HLB was categorized as the Candidatus Liberibacter asiaticus transmitted by the Asian citrus psyllid (Diaphorina citri). This was also reported in the south of Iran over 2000. Diagnosis of HLB disease can be difficult under field conditions when relying on visual surveys. This is due to its low concentration in its citrus hosts and the nonspecific nature of HLB symptoms, similarity of its symptoms to micronutrient deficiency such as zinc, magnesium, and iron and virus-like disease symptoms such as stubborn caused by Spiroplasma citri. Therefore, distinguishing causal agents of similar symptoms such as nutritional or stress related symptoms from HLB disease needs a robust procedure. Many detection methods have been used to detect Candidatus Liberibacter spp. including biological indexing (grafting and vector), electronic microscopy (EM), DNA probe specific to the bacterium, enzyme- linked Immunosorbent assay (ELISA). However, TEM methods are less practical, because ultra-thin sectioning is tedious and requires expensive equipment. Transmission tests are of limited value due to the latency and the long incubation period in insect and plant. Development of conventional polymerase chain reaction (PCR) test has great advantages of analyzing the bacterium at genetic level. The objective of this study was the detection of HLB in symptomatic citrus plants based on conventional polymerase chain reaction assay (PCR) in the south of Iran and the comparison of Iranian strains with other strains on the globe. In order to detect, identify and characterize the diseases, leaf and fruit samples were collected from some suspected sites situated in the southern Kerman. Samples were stored in plastic bags and transferred into the laboratory and conserved at low temperature (60C) before DNA extraction. Total DNA was extracted from symptomatic samples on the basis of the method/protocol of Murray and Thompson (1980) with minor modifications. Briefly, 5 to 10 symptomatic leaves were washed with sterile water and dried on paper. The leaf midrib tissue derived from field plants was cut out by sterile scalpel and CTAB buffer added with addition of B-mercaptoethanol. The extract was transferred to a new tube and incubated at 65 0c for 15min. The other steps were conducted according to the original protocol. Existence of pathogen in samples was confirmed using PCR with primers A2 / J5 and OI1 / OI2c. The PCR products were analyzed by gel electrophoresis using a 1% agarose in TBE buffer (Tris base, boric acid and 0.5M EDTA [pH 8.0]) and stained with ethidium bromide. Gel was visualized and analyzed by the GEL documentation.12 of 48 samples amplified with primers above and the disease was confirmed. By sequencing of PCR products with length of 1077 bp and comparison with the strains positive control, also production two fragments of 640 bp and 520 bp resulting from the digestion with XbaI PCR products, the agent of disease was found to be Candidatus Liberibacter asiaticus. The agent showed 100% homology with standard HLB Asiatic type. BLAST analysis showed that the nucleotide sequences obtained for the ribosomal protein (GenBank Accessions No. GN 049632) had 100% identity with sequences of ‘Ca. L. asiaticus’ from China (DQ431997), Taiwan (AB555707), Indonesia (AB480102), Florida (CP001677), and Brazil (AY91933). The Asian vector of HLB, Diaphorina citri was reported in 2000, therefore, the diseases might be distributed in other areas in the southern Iran. Thus, detection of HLB disease in young citrus plants is important to prevent a widespread outbreak of this disease. The results also showed that Iranian strain belongs to Asian type of Liberibacter and nominated Candidatus Liberibacter asiaticus. In the southern Iran, Diaphorina citri with high ability to spread the Candidatus Liberibacter asiaticus is found in most of citrus cultivating areas which implies a high risk of rapid dissemination. Therefore, the survey of the disease by an accurate and sensitive method is recommended for the disease detection in new areas and eradication of infected trees.

Abstract During years 2016-2017 surveys in apple orchards of Maragheh region, 73 samples of Gala and Red delicious cultivars with symptoms resembling to those caused by Apple dimple fruit viroid (ADFVd) were collected and subjected to two-step RT-PCR. Total RNA from stem bark and leaves of symptomatic plants showing dimple fruit and irregular yellow spots on fruit was extracted and full-length 306-307 base pairs genome of the isolates were amplified by using specific primer pairs. Ten amplicons with expected size were directly sequenced on both strands. Blast analysis and multiple sequence alignment of the sequences with those full-length ADFVd sequences deposited in GenBank, showed identities ranging from 74.4-100%. Iranian ADFVd isolates had the highest and lowest identity with two Italian isolates, accession Nos. EF088662 and KF788291, respectively. The region between CCR and TR domains was the most divergent. In phylogenetic analysis by maximum likelihood method, all Iranian isolates were clustered together with the Italian isolates and variants.

The plant pathogen Phytophthora melonis is morphologically similar to some other non-papillate Phytophthora spp. especially P. drechsleri and therefore it is difficult to discriminate these convergent taxa. This study was performed to design specific primers based on nuclear and cytoplasmic genome, examine their specificity against other convergent species, and optimize the specific primers’ conditions to detect P. melonis. Nine nuclear and four cytoplasmic genes were appropriate to design thirteen specific primers based on their nucleotide polymorphism. PCR conditions were optimized. Phytophthora melonis were detected by specific primers in inoculated plants such as cucumber, watermelon, melon, sugar beet and pistachio. All specific primers detected pathogen in 1:100 (pathogen: soil) inoculated soil.Up to 10 zoospores per milliliter were detected using nested polymerase chain reaction. ITS-MF1 and ITS-MR2 (ITS-M2 set, from internal transcribed spacers of rRNA gene) were selected as the most efficient primers based on their specificity and sensitivity. The optimized annealing temperature for this primer set was 68 °C. It seemed that nested PCR by ITS-M2 primer set together withthe universal primers ITS6 and ITS4 as external primers is at least 106 times more sensitive than simple PCR. Multiplex polymerase chain reaction simultaneously detected the three cucurbits’ pathogens including P. melonis, P. nicotianae and P. drechsleri. This study showed that the designed primers could be effective tools for detection of P. melonis isolates from infected tissues, and infested water and soil.

Fig badnavirus-1, a species of the genus Badnavirus in the family Caulimoviridae, is one the most prevalent viruses associated with the fig mosaic disease through in fig plantations worldwide. During the growing seasons of the year 2012-2013, a total of 392 asymptomatic and symptomatic leaf samples were collected from different fig orchards located in 9 provinces of Iran. Using the DIBA serological test and universal serum, samples were tested for the presence of fig mosaic disease. Following total DNA extraction from infected samples, an amplicon with a size about 1055bp was amplified using specific primer for FBV-1 protease gene. RFLP test was conducted with the PCR products of the FBV-1 infected samples and EcoRI enzyme to assess the genetic variation of infected isolates. Protease gene of nine representative FBV-1 samples from different provinces were sequenced and submitted in GenBank under the accession numbers KU314932-40. Blast analysis showed 98-99% identity between the Iranian isolates at the nucleotide and amino acid levels. Phylogenetic analysis on the basis of the nucleotide sequences of studied isolates categorized them into two different groups in which Iranian isolates grouped together in a separate group distinct from American isolate. Phylogeny on the basis of the amino acid sequence revealed a different phylogenetic tree. Totally, results of this study indicated the presence of different FBV-1 populations in Iran.

Phytoplasmas are phytopathogenic bacteria without cell walls that can be found in plant phloem, have been found associated with numerous diseases of plants worldwide. A sensitive and precise diagnostic test for detection of phytoplasma-infected plant is critical to avoid using infected planting material and dispersal of these agents. The detection of phytoplasma from plant tissues by PCR, requires using DNA extraction methods that extract high level phytoplasmas DNA with less plant inhibitors. Usually Phytoplasma diagnostics have been based on the 16S rRNA gene and the 16S–23S rRNA spacer region because universal primers that design for replication these regions could detect different groups of phytoplasmas but diagnostics based on these primers can be problematic, with occasional false positives, through amplification of some bacterial genomes that might be present in a plant sample. These primers also have sequence homology to chloroplasts and plastids and increase the risk of false positives so it is important to guard against false negatives and positive during such detection techniques. Health and infected Lime samples with Candidatus phytoplasma aurantifolia were used respectively as negative and positive samples. DNA was extracted by the CTAB methods, SDS method, Fermentas DNA extraction kit, column based Method (Genet Bio genomic DNA isolation kit) and compared to remove inhibitors and reduced false negative reactions in nested PCR detection method. DNA samples were tested for phytoplasma infection by direct PCR using the universal phytoplasma primer pair P1/P7 and nested PCR using primer pairs P1/P7-R16F2n/R16R2 and P1/P7-fU5/rU3. The PCR products were sequenced and subsequent analysis using GenBank database information at the national center for biotechnology was employed. Comparison of different DNA extraction methods indicated using suitable method can significantly reduce false negative reaction, but even in successful column-based DNA extraction method, false negative reactions were reported that were due to low phytoplasma concentration and irregular distribution within host tissues or could be caused by inhibitor presence in DNA samples. Based on results of sequencing, false positives were obtained sporadically, using primer pairs combination P1/P7- R16F2n/R16R2 that may be arising from cross over contamination or sequence homology with plant genome, so some primers can react probably with sequences of plant genome and false positives could be observed. Since false positives are also a major problem in PCR protocols, especially in nested PCR so single tube nested PCR (STNP) was optimized to avoid false positive reaction but regardless advantages of this method such as facility, cost and time effective and ability to detect low concentration of pathogen, false positive reactions were observed in a few samples. The advantage of STNP is that tubes do not have to be opened, so the risk of contamination minimized. PCR-based techniques for phytoplasma detection, appears to be the method of choice because of their high sensitivity and specificity. Using suitable method for extraction of DNA from infected plant tissues are getting more critical for precise detection through increasing DNA quality and quantity. In the other hand confirmation of phytoplasma presence must be accomplished at least by RFLP analyses or different primer pair combination to avoid false positive detection.

Lily (Lilium spp.) from Liliaceae familiy is considered as one of the most important ornamental plants with high economic value. Virus diseases are shown to be as main limiting factor in lily production. During a survey in 2015, a total of 76 symptomatic samples showing mosaic, deformation, chlorosis, reduced growth, necrosis and decline were collected from lily growing greenhouses in Thehran province. Samples were tested for Cucumber mosaic virus (CMV) and Arabis mosaic virus (ArMV) using ELISA, biological and molecular methods. ELISA results showed that 45 and 3 samples were infected with CMV and ArMV, and 5 samples were co-infected with the both viruses. The virus infections were confirmed using biological inoculation tests. CMV and ArMV infection were also tested by RT-PCR method using specific primers which resulted to amplification of 1100 and 690 bp DNA fragments, respectively. CMV lily isolates were confirmed to belong to CMV subgroup I using specific primers in RT-PCR assay. Presence of Lily symptomless virus –LSV and Lily mottle virus-LMoV were tested by RT-PCR assay using specific primers which resulted in amplification of the expected 650 bp DNA product corresponding to CP gene region of LMoV, but there was not any DNA amplifications related to LSV. This is the first report on occurrence of ArMV, CMV and LMoV infections on lily in Iran. Detection of virus infection sources using the optimized methods in this study is a crucial practical approach for virus management in healthy lily bulb production in the country.

One of the most significant and destructive grapevine diseases is Esca which is caused by several fungal species. Trunk disease causes stunted growth، reduced yield، and ultimately، vine death. For this reason، during the summer 2011، infected samples were collected from Bojnourd (North Khorasan province) vineyards. Infected trunk samples were acquired that they were showing white and brown rots، dark brown spots and also samples without apparent symptoms. The aims of this work were، firstly، to apply species –specific primers for detecting fungal agents associated with Esca disease of grapevine; and comparing this method to the classical ones، and secondly، to detect frequency of each pathogenic agent using molecular and classical methods. In molecular method، species-specific primers which designed based on ITs region of rDNA were used to detect fungal agents of the Esca disease. They yielded a single amplicon of 550 bp، 360bp and 415 bp for F. mediterranea، Pa. chlamydospora and Phaeoacremonium spp. respectively. Results showed that the maximum frequency of incidence was related to P. chamydospora and Phaeoacremonium spp was in second. The lowest incidence frequency rate was related to F. mediterranea which was observed only in sample with white rot symptoms. Primers defined here can be used in the nursery sanitation program to produce plant free of Esca disease.

In order to detect Onion yellow dwarf virus in Khorasan province in Iran، onion samples from major onion growing regions where collected in spring and summer 2010. To determine the distribution of the virus، samples were randomly taken from onion fields in vicinity of Mashhad، Chenaran، Quchan، Torbate Heydarye، Neyshabur، Bardaskan، Gonabad، Fariman، Faruj، Shirvan، Bojnourd and Ash khane have been tested by DAS- ELISA method. Leaf samples showing mosaic، yellowing، rolling symptoms and complexity associated with dwarf onion bulbs glands were collected. Infection of samples with Onion yellow dwarf virus were confirmed using DAS-ELISA and RT-PCR tests. Total RNA was extracted from infected samples using RNX ™ (plus) kit. To perform RT-PCR test cDNA was made and then PCR test was carried out. 1. 5% Agarose gel electrophoresis of PCR products resulted in a fragment of 291 bp from CP of the virus. Amplified fragment were then sequenced. The virulence of the virus in greenhouse on three Chenopodium species (Chenopodium album، C. quinoa، C. amaranticolor) was confirmed. The results showed that onion field in Chenaran، Gonabad and Neyshabour infected with Onion yellow dwarf virus but no infection in other places.

Spiroplasma citri, the causal agent of stubborn disease has become one of the destructive pathogen of citrus industry in Iran. One of the most important strategies for control of stubborn disease is using S. citri-free mother trees, propagative material and citrus nurseries and disease avoidance in new orchards. In this way, the efficiency of the standardized ELISA with poly clonal antibody and PCR protocol with three different primer pairs was assessed. The results revealed that ELISA was not able to detect low titre of infection (in symptomless samples) so using ELISA techniques in certification system was not recommended and has significant problem such as false negative reaction. PCR method and specially P89f/r primers in compare with two other primers P58-1f/5r and P58-6f/4r was able to detect low titre of north and south strains of pathogen in moderate seasons. Out of a total 350 samples screened for presence of S. citri by the PCR method only 4/6 % showing positive results in symptomless samples.

Armillaria mellea the causal agent of root and crown rot of trees has a universal distribution and causes extensive economic disease on a broad host range of trees in gardens, forests and urban environments. The pathogen can survive under the bark as inoculum and there is no effective control method against the disease, so pathogen detection from soil and wood is important to predict disease severity and prevent disease dispersal from one tree to another. To achieve a rapid and sensitive detection method for A. mellea in soil and wood, sampling was performed from fruiting bodies, soil and wood and after isolation of pathogen on culture media, comparison was done among different detection methods using a semi-selective medium, baiting and molecular methods. Inoculum was prepared for two isolates of A. mellea and was inoculated to soil. Pathogen detection was done with soil direct culture method, baiting and nested PCR simultaneously. The culture medium was not effective to detect pathogen in long-term period. In baiting method, Pelargonium hortorum was used as a baiting plant that required long time to detect pathogen. The results revealed that nested PCR is an efficient method for detection of A. mellea.

Phytophthora inundata has been recently described and depicted as a ‘difficult’ taxon for identification due to its morphology and unusually high upper temperature limit for growth which has been mistaken for other non-papillate and high temperature tolerant plant pathogenic Phytophthora species. A simple as well as a nested-PCR based method was developed for the identification of P. inundata. A collection of isolates from different hosts representing diversity of species were examined for unique regions of coding as well as non-coding gene sequences. Based on internal transcribed spacers 1, 2 and 5.8S gene of rDNA (ITS), heat shock protein 90 gene (HSP), triosephosphate isomerase/glyceraldehyde-3-phosphate dehydrogenase fusion protein (TIG), and 60S ribosomal protein L10 gene (RPL) ten PCR primers specific for P. inundata were designed. Annealing temperatures and extension times were optimized for each set of primers for maximum specificity and efficiency. To evaluate the specificity of the method, 28 morphologically and molecularly characterized Phytophthora species were tested. In most cases neither set of primers amplified purified DNA from these non-homologous Phytophthora species. Analysis showed that the best candidate for identification of P. inundata isolates is ITS-I1 set which is a combination of ITS-IF1 and ITS-IR1. The optimized annealing temperature for this set was 69 °C and the best extension time was 40 sec. It seems that nested-PCR by ITS-I1 set together with universal ITS6 and ITS4 as external primers is at least 50 times more sensitive than conventional PCR.