Genetic diversity of hop stunt viroid from symptomatic and asymptomatic citrus trees in Iran

Hop stunt viroid as the causal agent of cachexia disease has detected from citrus trees in different areas in Iran. Although cachexia has not been reported as a decline disease for citrus trees, it can impair crop quality and reduce plant yields. This study was undertaken to molecularly detect HSVd among different commercial citrus cultivars and determine genetic diversity of this viroid in Mazandaran province of Iran. Sampling was performed from symptomatic and symptomless citrus cultivars in Mazandaran province. HSVd specific primers were used for molecular detection. SSCP and sequencing were applied to assay HSVd genetic diversity. Results showed the detection of HSVd in all symptomatic Satsuma (25 out of 25), Clementine (25 out of 25), sweet lime (20 out of 20) and sweet orange cv. Valencia (7 out of 7), as well as, 31% (14 out of 22), 100% (12 out of 12) and 33% (5 out of 15) of page mandarin, lemon and grapefruit trees, respectively. 10 different HSVd genomes were identified by sequencing the SSCP profiles among which HSVd‐IR1 had the most frequency.     

Occurrence of Hop Stunt Viroid in Mulberry (Morus alba) in Lebanon and Italy

The presence of Hop stunt viroid (HSVd) was detected using RT‐PCR and Northern blot hybridization in five of 60 samples from symptomless mulberry trees (Morus alba) collected in Italian and Lebanese orchards in July 2010. Infection levels were c. 10% in Lebanese and 8% in Italian samples. Nucleotide alignments showed that sequences of the mulberry HSVd isolates shared 95–96% identity with those of the same viroid occurring elsewhere. In a phylogenetic tree, mulberry HSVd isolates clustered together with those of HSVd‐citrus, regardless of their geographical origin. This is the first report of infection in mulberry trees by HSVd.     

Molecular,Biological and Phylogenetic Analysis of Chinese Isolates of Hop stunt viroid Associated with Citrus Cachexia Disease

Citrus cachexia is an economically important disease of citrus hosts caused by specific variants of Hop stunt viroid (HSVd) that are usually referred to as Citrus cachexia viroid (CCaVd). Eight cachexia‐associated HSVd isolates were collected from six citrus growing areas of China, where citrus cachexia had not been reported previously. Forty‐seven independent cDNA clones were used for genetic diversity and phylogenetic analysis. There were no sequence variant‐cultivar correlation and no distinct regional specificity among or within the cachexia‐associated HSVd populations analysed. Three clusters consisting of three major HSVd variants were identified by phylogenetic analysis, suggesting that most Chinese isolates contain a mixture of cachexia and non‐cachexia variants. Biological properties of eight CCaVd isolates were determined by inoculating Parson’s Special mandarin (Citrus reticulata). Our results suggest that the interactions between CCaVd and non‐CCaVd variants might play an important role in suppressing cachexia symptom expression.     

Cost–benefit analysis of pistachio twig borer,Kermania pistaciella Amsel (Lepidoptera: Oinophylidae) chemical control

Pistachio twig borer is a major pest of pistachio orchards in Iran. In order to reduce the pest damage, a conventional pesticide application is used annually. However, it is not known how much damage is caused by this pest. To determine the potential damage of Kermania pistaciella, and to qualify how efficient chemical control is in reduction of the pest damage, an experiment was conducted at two pistachio orchards in Kerman province (Kerman and Sirjan). A randomised complete block design, with two treatments – chemical control (a mixture of endosulfan EC 35 and volk, 2.5:5 l/ha) and check with five replications were performed. To estimate the pest damage, 25 randomly selected trees were chosen then the total fruit bunches of each tree were checked for infestation to the pest. The infestation percentage in Kerman was greater than that for Sirjan region (10.3% vs. 5.9%). When treatments were compared, the infestation rate of check plots were significantly more in comparison of treated plots in Kerman (10.3% vs. 3.6%); however, there was no significant difference between treatments in Sirjan (5.9% vs. 3.1%). The efficiency of chemical control was 60.7% and 48.7% in Kerman and Sirjan, respectively. There was not definite correlation between the number of capture and the infestation rate (r = 0.442). Cost–benefit ratio calculation was less than 1 (Kerman (?0.89) and Sirjan (?0.94)) and it means that chemical control at this damage percentage is not economic.     

Detection of Aflatoxin in Aspergillus Species Isolated from Pistachio in Iran

To estimate the incidence contamination of fresh pistachio nuts by aflatoxigenic fungi in Iran, nut samples were collected from pistachio orchards in Kerman, Rafsanjan and Isfahan regions. Out of the 200 Aspergillus isolates obtained, 11 species were identified as A. alliaceous, A. candidus, A. flavus, A. niger, A. niveus, A. ochraceus, A. parasiticus, A. tamari, A. terreus, A. unguis and A. wentii. For detection of aflatoxin production ability of the isolates, three target genes, namely aflR, aflJ, and omtB, used in PCR amplification. In all the examined cases, the degenerate primer designed for amplification of omtB gene, named omtBII, was able to amplify an expected 611 bp fragment in aflatoxigenic isolates in this study and yielded the same result as those obtained from TLC analysis and fluorescence ability by application of methylated β‐cyclodextrin in culture media. Using this procedure the significant incidence of aflatoxin‐producing aspergilli was confirmed in pistachio nuts produced in different regions of Iran. The results indicated that PCR method described here, in combination with fluorescence assay, is a reliable and simple confirmatory test for monitoring pistachio nuts contaminated with aflatoxinogenic aspergilli.     

Molecular identification of 16S rII phytoplasma group in commercial pistachio cultivars in Iran

Evaluation of phytoplasmas infection was conducted in the pistachio-growing areas of Iran (Rafsanjan in Kerman province) in early autumn of 2011. A total of 30 pistachio trees collected from a pistachio orchard in Rafsanjan showing Psylla damage symptoms and 10 samples with different abnormal symptoms from miscellaneous orchards were tested for the presence of phytoplasma. By using nested PCR with primer pairs P1/P7 and internal primer sets R16F2N/R16R2 and fU3-rU5, amplified fragment of expected size was observed in some trees with deformation and yellowing symptoms. On the basis of nucleotide sequence analysis of 16S?rDNA amplified by PCR, this phytoplasma was classified in group 16S?rII. In addition, we observed false positive reaction in three trees of Ahmadaghaei cultivar by using primer sets R16F2N/R16R2 and sequence analysis of ~1250bp PCR product indicated that amplified fragment was related to Schinus terebinthifolius; a species of flowering plant in the Anacardiaceae family.     

Toxic aspergilli from pistachio nuts

Pistachio nut samples taken during various stages of development from orchards in Iran, showed that contamination with fungi occurred mainly during the later stages of nut development. Members of the genera Aspergillus and Penicillium occurred most frequently. Of the Aspergilli, the species A. niger, A. flavus and A. fischeri var. spinosus occurred most frequently, followed by A. terreus, A. tamarii and A. nidulans. Twenty-two isolates comprising 13 species were tested for toxicity to ducklings. Isolates of known toxic fungi included A. flavus, A. niger, A. parasiticus, A. ochraceus, A. versicolor, A. nidulans and A. terreus. The toxicity of A. fischeri var. spinosus is reported. Chemical analysis showed that all isolates of A. flavus and A. parasiticus produced aflatoxin B₁, the isolates of A. versicolor and A. nidulans produced sterigmatocystin while the toxic isolate of A. ochraceus did not produce ochratoxins.Toxic fungi have been shown to occur in a variety of nuts (4), (5), (11), (12), (13), (15), (18), (20), (21). Aflatoxin contamination of pistachio nuts has been reported and has in the past led to the rejection of consignments of Iranian pistachio nuts (1). In Iran, pistachio nuts are produced mainly in the south eastern provinces (Kerman & Zahedan) and to a limited extent in the Northern part (Kazvin & Damghan). In 1975 it was estimated (7) that there were some 24 million pistachio trees in Iran, of which 60% were situated in Rafsanjan, Kerman (Table 1). Economic considerations as well as the potential health hazard posed by aflatoxin-contaminated nuts, prompted the University of Isfahan to initiate a study of various aspects of the mycotoxin problem in pistachio nuts.     

Collophora hispanica,a New Pathogen and Potential Threat to the Almond Industry in Iran

Trunk diseases are potential threats for almond productivity and longevity worldwide, including Iran. In a recent survey on fungal species associated with trunk diseases of almonds in north‐western Iran, Collophora isolates (tentatively identified as Collophora hispanica) were recovered with high frequency from wood samples with internal necrosis and brown to black vascular streaking of almond trees showing symptoms of decline. However, the pathogenic potential of Collophora isolates on almond trees in Iran remains unproven. In this study, the identity of the isolates was further confirmed as C. hispanica based on a combination of morphological data and sequence data of ITS‐rDNA region, and pathogenicity of C. hispanica isolates on almond was evaluated using excised shoot method and in greenhouse experiments. Collophora hispanica isolates induced lesions statistically different from the control, in both excised shoot method and greenhouse assays. Significant differences were observed among the isolates in the length of the lesion induced on wood. Collophora hispanica should be considered as the main trunk pathogens of almond trees in north‐western region of Iran. The distribution and host range of this new pathogen on almond remains to be studied.     

Cultivated Grapevines Represent a Symptomless Reservoir for the Transmission of Hop Stunt Viroid to Hop Crops: 15 Years of Evolutionary Analysis

Hop stunt was a mysterious disorder that first emerged in the 1940s in commercial hops in Japan. To investigate the origin of this disorder, we infected hops with natural Hop stunt viroid (HpSVd) isolates derived from four host species (hop, grapevine, plum and citrus), which except for hop represent possible sources of the ancestral viroid. These plants were maintained for 15 years, then analyzed the HpSVd variants present. Here we show that the variant originally found in cultivated grapevines gave rise to various combinations of mutations at positions 25, 26, 54, 193, and 281. However, upon prolonged infection, these variants underwent convergent evolution resulting in a limited number of adapted mutants. Some of them showed nucleotide sequences identical to those currently responsible for hop stunt epidemics in commercial hops in Japan, China, and the United States. Therefore, these results indicate that we have successfully reproduced the original process by which a natural HpSVd variant naturally introduced into cultivated hops was able to mutate into the HpSVd variants that are currently present in commercial hops. Furthermore, and importantly, we have identified cultivated grapevines as a symptomless reservoir in which HSVd can evolve and be transmitted to hop crops to cause epidemics.     

High‐throughput sequencing of Hop stunt viroid‐derived small RNAs from cucumber leaves and phloem

Small RNA (sRNA)‐guided processes, referred to as RNA silencing, regulate endogenous and exogenous gene expression. In plants and some animals, these processes are noncell autonomous and can operate beyond the site of initiation. Viroids, the smallest self‐replicating plant pathogens known, are inducers, targets and evaders of this regulatory mechanism and, consequently, the presence of viroid‐derived sRNAs (vd‐sRNAs) is usually associated with viroid infection. However, the pathways involved in the biogenesis of vd‐sRNAs are largely unknown. Here, we analyse, by high‐throughput pyrosequencing, the profiling of the Hop stunt viroid (HSVd) vd‐sRNAs recovered from the leaves and phloem of infected cucumber (Cucumis sativus) plants. HSVd vd‐sRNAs are mostly 21 and 22 nucleotides in length and derived equally from plus and minus HSVd RNA strands. The widespread distribution of vd‐sRNAs across the genome reveals that the totality of the HSVd RNA genome contributes to the formation of vd‐sRNAs. Our sequence data suggest that viroid‐derived double‐stranded RNA functions as one of the main precursors of vd‐sRNAs. Remarkably, phloem vd‐sRNAs accumulated preferentially as 22‐nucleotide species with a consensus sequence over‐represented. This bias in size and sequence in the HSVd vd‐sRNA population recovered from phloem exudate suggests the existence of a selective trafficking of vd‐sRNAs to the phloem tissue of infected cucumber plants.     

Simultaneous Detection of Three Viroid Species Infecting Hops in China by Multiplex RT‐PCR

We have developed a multiplex RT‐PCR protocol for the simultaneous detection of three viroids in three different genera that infect hops: Hop latent viroid (HLVd; Cocadviroid), Hop stunt viroid (HSVd; Hostuviroid) and Apple fruit crinkle viroid (AFCVd; Apscaviroid). The method was validated by testing 175 hop samples collected from the Xinjiang autonomous region of China. All samples were found to be positive for HLVd but negative for AFCVd, confirming the widespread or even ubiquitous infection of HLVd and the low incidence of AFCVd in hops in China. In addition, HSVd was detected in 22.86% of the samples tested. This rapid and reliable multiplex RT‐PCR assay provides an effective method for detection of three important viroid species in large‐scale surveys for disease management in hops.     

A new report of pre-harvest ear rot of corn caused by Geotrichum candidum from Iran

In summer of 2004, samples of husk looseness ear of corn (Zea mays) (cv. 700-Karaj) were collected from corn fields in Ali-Abad (Jiroft region), Kerman province, Southeastern Iran, for diagnosis of an unusual ear decay. A fungus was isolated from the rotting kernels and subsequently identified as Geotrichum candidum. The fungal pathogen was found to be closely related to G. citri-aurantii (citrus race) based on morphological, physiological and pathogenicity properties. The fungal pathogenicity test was demonstrated by fulfilling Koch's postulates. The pathogen caused rot disease on husk looseness corn kernels in soft-dough stage of ripening. The fungus was also pathogenic on ripe lemon and green and ripe tomato fruits. Fungal isolates of corn were compared to isolates from soft-rotten potato tubers. These two groups of isolates were highly similar on the basis of their morphological, biochemical and pathogenicity characteristics. To our knowledge, this is the first known report of corn ear rot caused by G. candidum in the world.     

Detection of multiple sequence variants of Grapevine rupestris stem pitting‐associated virus using primers targeting the polymerase domain and partial genome sequencing of a novel variant

Grapevine rupestris stem pitting‐associated virus (GRSPaV) is a member of the genus Foveavirus within the new family Betaflexiviridae. GRSPaV is distributed among grapevines worldwide and is implicated in the disease rupestris stem pitting (RSP) of the rugose wood complex and two other disorders. GRSPaV is composed of a wide range of sequence variants, and so far, the complete genomes of five sequence variants have been sequenced. Quick and reliable detection of different GRSPaV variants is a critical step in the elimination and control of GRSPaV. Previously, primers designed from various genomic regions have been used in RT‐PCR for the detection of GRSPaV variants. The efficiency of RT‐PCR varied widely depending on the spectrum of the primers that were used. In this study, we designed a pair of degenerate primers based on the consensus sequence of the genomic region encoding the highly conserved RNA‐dependent RNA polymerase domain from five reference isolates of GRSPaV for which the genome sequence are available. We demonstrate that this set of primers is comparable, if not superior, to the broad‐spectrum primers RSP13&14 in detecting multiple GRSPaV variants. Using these degenerate primers, we identified two new and distinct sequence variants. The 3′ terminal genomic region of one of the new variants, GRSPaV‐ML, spanning the 3′ part of ORF1, through the entire open reading frames 2–4, and the 5′ region of ORF5 were sequenced. Sequence comparison demonstrates that GRSPaV‐ML is distinct from each of the five reference isolates.     

Replication of in vitro constructed viroid mutants: location of the pathogenicity-modulating domain of citrus exocortis viroid

Sequence variants from field isolates of citrus exocortis viroid (CEV) that cause either mild or severe symptoms on tomato plants have previously been classified into two groups, A and B. These groups differ primarily in two domains, P_L and P_R, of the proposed native structure. Infectivity studies with full-length cDNA clones of variants from each class have now directly confirmed the original correlation between Class A sequences and the severe phenotype and between Class B sequences and the mild phenotype. Direct evidence for this correlation could only be obtained by using individual sequence variants since field isolates of CEV have been shown to contain a mixture of RNA species. The construction and infectivity of chimaeric cDNA clones derived from mild and severe sequence variants of CEV has demonstrated that novel, infectious viroid molecules can be generated in vitro, and that P_L is the pathogenicity-modulating domain. The role of the P_R domain is not known but infectivity experiments with one chimaeric cDNA clone suggest that it may influence the efficiency of the infection or replication process of the viroid in the plant.     

Behaviour of Apricot Cultivars Against Hop Stunt Viroid

The apricot species is susceptible to infection by different viruses and viroids including the Hop stunt viroid (HSVd), widely distributed around the world and described as the causal agent associated with fruit degeneration disease in apricot. However, to date, there are no ‘ad hoc’ studies about the resistance or susceptibility of apricot cultivars to this viroid. In this study, we tested the resistance/susceptibility to HSVd of 26 Mediterranean and North American apricot cultivars in controlled greenhouse conditions. All apricot cultivars assayed were infected by HSVd, showing a different range of susceptibility. As no sources of resistance among the apricot cultivars evaluated have been detected yet, it is necessary to continue the search for such sources to be included in apricot breeding programmes. In addition, the high level of susceptibility found highlights the importance of identifying this viroid in routine tests performed by nurseries and plant protection services.     

Uneven Distribution of Mating‐Type Alleles Among Togninia minima Isolates,One of the Causal Agents of Leaf Stripe Disease on Grapevines in Northwest Iran

Togninia minima is the main fungal species associated with grapevine leaf stripe disease worldwide. This species is mainly known from its asexual state in nature; nevertheless, a biallelic heterothallic mating strategy has been confirmed for this species based on in vitro crossing studies. There are no data available on the incidence of an active sexual cycle within the populations of this species in many grapevine‐producing countries as well as Iran. The possibility of a clandestine sexual cycle within the Iranian isolates of T. minima was evaluated by analysing the distribution and frequency of the mating‐type alleles on a microspatial and a macrogeographical scales. Towards this aim, a total of 90 T. minima isolates were recovered from grapevines with esca disease from the vineyards in north and north‐western Iran. A multiplex PCR method previously designed by authors was applied for simultaneous identification and determination of the mating‐type alleles in T. minima populations. The results on the screening of mating‐type alleles using multiplex PCR method revealed the mating‐type identity of 77 isolates as Mat1‐2 and 23 isolates as Mat1‐1. Our results showed that both Mat1‐1 and Mat1‐2 isolates are present in a single vineyard and even on single vines. The distribution of mating‐type alleles in the sampled area skewed from the 1 : 1 ratio (77 : 23); however, co‐occurrence of both mating types in a single vineyard and even on single vines is suggestive for the presence of an active sexual cycle for T. minima in north‐western Iran.