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.

Introduction: APPLE (Malus spp. ) is one of the most economically important fruit crop worldwide. This crop is highly affected by various virus infections, leading its considerable devastation and eventually results in yield loss in the whole world. Among effective viruses, APPLE stem grooving virus (ASGV), APPLE stem pitting virus (ASPV), and APPLE chlorotic leaf spot virus (ACLSV) which have been firstly characterized in APPLE (Malus domestica), play important roles in altering the plant defense mechanism, leading a low performance. According to the references, heat treatments can reduce the movement of virus particles into the apical meristem through inhibiting viral RNA synthesis, so high temperature over a long period is an efficient method for virus elimination. In additions, meristem culture is also a common method to eradicate viruses from horticultural plants. This study was done to produce virus-free (ACLSV, ASPV, ASGV) APPLE (Malus pumila Mill. ) plantlets. Materials and Methods: The effect of different thermotherapy duration (0, 7, 14 and 21 days at 38 ° C) and the sizes of apical meristems for meristem culture(less than 0. 2 mm, between 0. 2 and 0. 7 mm and larger than 0. 7 mm) was assessed on virus eradication. Our plant material was a genotype of red flesh APPLE belongs to Budagovsky Bud. 9 (Malus+pumila) "Niedzwetzkyana" of Rosaceae family from Shahroud, that has high levels of important phytochemicals like antioxidants, flavonoids and anthocyanins in its cortex, which in addition to attractiveness, creating special healing properties in some disease. At first the presence of ACLSV, ASGV and ASPV were checked in mother samples by ELISA and RT-PCR methods. Then we performed the thermotherapy treatments and later meristems were cultivated and grown in vitro. Regenerated shoots from meristem were tested by ELISA and RT-PCR methods for all three viruses. Samples that were diagnosed virus-free by both techniques, were proliferated, rooted and transferred into the pots to be used for later propagation and establishment of the mother orchard. Results and Discussion ELISA results for the presence or absence of ACLSV, ASGV and ASPV were indeterminate; they were neither negative nor positive for each virus, indicating ELISA was not an accurate method to study virus infections in our samples. Therefore, careful examination of initial infection of samples was performed by RT-PCR. Examining mother samples by RT-PCR showed that all samples were infected by ASG, ASP and ACLS viruses. Results of RT-PCR testing suggested that the number of days in thermotherapy and the size of meristem had a significant effect (P<0. 01) on the ACLSV, ASGV and ASPV elimination. Increasing in the duration of the thermotherapy decreased the survival rate of the explants and it was difficult to acquire virus-free explants after 21 days treatments, because all the explants were destroyed in this treatment. The relation between the size of meristem and its survival has been examined in different studies, whose results are consistent with the present research and confirms that increasing the size of the meristem and the number of leaf primordium, will increase the vitality. However, it should be noted that larger meristems are more susceptible to viral contamination. In the previous study, the percentage of ASGV and ACLSV elimination was significantly influenced by the size of the meristem and the duration of the thermotherapy. In the same study, ACLSV, ASGV and ASPV could not be detected in plants grown from meristems smaller than 1 mm after 35 days of thermotherapy. They also showed that virus eradication was happened after shorter period of thermotherapy if smaller meristems were cultured (0. 7 mm). The results of our study were consistent with these observations; ACLSV, ASGV and ASPV appeared to be eliminated more frequently from the smallest meristem treatment (smaller than 0. 2 mm) compared to the other treatments (0. 2-0. 7 mm and larger than 0. 7 mm). Elimination rates of ACLSV, ASGV and ASPV were different in each case and ASPV-free frequency obtained in regenerated shoots treatment was higher than other viruses (ASGV and ACLSV). This observation was similar to the results of some previous studies. It could be due to differences in the morphology of the viruses and transmission factors in plant cells that affect viruses, which has been investigated and confirmed in previous studies. Meristem culture alone, without other treatments, had very low efficiency in virus elimination from explants. The best results of virus eradication from APPLE explants was obtained after 14 days of thermotherapy, that we had 44. 44%, 11. 11% and 88. 89% of virus-free samples from ACLSV, ASGV and ASPV, respectively. Generally, our results showed that by increasing the thermotherapy duration, the elimination of all three types of viruses was increased. Many researchers also found that thermotherapy is a very effective treatment in virus eradication. Conclusion: Results showed that increasing the duration of thermotherapy (14 days) and reducing the size of cultivated meristem (smaller than 0. 2mm) increased the elimination rate of all three viruses (ACLSV، ASPV، ASGV) from APPLE explants.

The present study aims at producing healthy primary pear plants for propagation and establishing a high health maternal orchard, for which an experiment was conducted in 2014 at Agriculture Biotechnology Research Institute of Iran (ABRII), trying to evaluate the effectiveness of chemotherapy treatments (Ribavirin 0, 20, 40, and 80 mg/l) as well as the sizes of apical meristems culturing (below 0. 2 mm, between 0. 2 and 0. 7 mm, and above 0. 7 mm) on eradication of some virus strains (ACLSV, ASPV, ASGV) from seven pear cultivars ("Abate Fetel", "Beiruti", "Coscia", "Dargazi", "Louise Bonne", "Mellina", and "Spadona"). Initially, the maternal samples are tested for ACLSV, ASGV, and ASPV by RT-PCR and all explants are infected with all three viruses, with the exception of Abate Fetel and Beirut which have been free from ASPV. The chemotherapy treatments and meristems cultivation take place in vitro, with the regenerated shoots from meristem tested by RT-PCR for all three viruses. Results show that increasing the concentration of ribavirin and reducing the size of cultivated meristem are effective on elimination of all three viruses from the explants. Also, the responses from the studied cultivars and virus varieties have varied among this experiment's treatments. Samples, diagnosed as virus free by means of RT-PCR, are then proliferated, rooted, and transferred into pots to be used for later propagation along with establishment of the maternal orchard.

APPLE (Malus domestica Borkh) is one of the most valuable agricultural products in the Rosaceae family because of its high nutritional and economic value. Powdery mildew of APPLE which is caused by Podosphaera leucotricha, is one of the primary fungal diseases with an extensively negative impact on the quality and quantity of APPLE production. In recent years, the use of resistant cultivars for controlling powdery mildew of APPLE has been considered. For this purpose, the candidate resistance genes Pl1, Pld, Plbj, and RGA (Resistance Gene Analogs) were detected in 47 genotypes of wild APPLEs collected from West Azerbaijan, Golestan, Fars, Chaharmahal va Bakhtiari and Isfahan provinces. The presence of NBS-LRR conserved domains and P-loop NTPase motif in NBS-LRR-like protein, which encode by the RGA in N-terminal, were confirmed in wild APPLE (Malus orientalis) genotypes of Iran. Pl1 resistance gene was detected in all collected samples in this research except one. Pld gene just was observed in three out of the 47 samples, and the Plbj resistance gene was detected in just 13 samples of all wild genotypes collected from cold areas of Azerbaijan and Isfahan provinces,the sequences of this gene was introduced to the Global database. Detection of resistance genes and RGAs can help in developing resistant APPLE varieties in future.

Patulin is a mycotoxin produced by a variey of mold species. In this study, a total of 70 samples consisting of 35 APPLE leathers and 35 APPLE juice samples was randomly obtained from different supermarkets of Shahrekord. Using HPLC-DAD technique and in accordance with the Iranian National Standard method, the samples were analyzed for patulin contamination. According to the results, patulin was detected in 14 (40%) of APPLE leather and 7 (20%) of the APPLE juice samples. Mean values of patulin concentrations in the APPLE leathert and APPLE juice samples were estimated as 34.9±26.3 and 26±19.7 m/kg, respectively. Although the mean concentration of patulin was lower than the Iranian/European approved limit (50 mg/kg), patulin contamination level was found higher than the maximum approved limit in 5.7% and 2.9% of the the APPLE leather and APPLE juices samples, respectively. Therefore, to minimize the contamination of patulin in theses products, the necessary practices should be conducted.

Relative resistance of 58 local APPLE genotypes to Erwinia amylovora  (Burrill.) Winslow et al., the causal agent of fire blight was studied. Bacterial strains were collected from pome fruit orchards of Orumieh, Karaj, Mashhad, Shahrud, Qazvin and Sanandaj cities and identified using phenotypical and molecular tests. A mixture of seven strains was used as inoculum.  Evaluation was performed using shoot tip inoculation method nuder glasshouse condition on one-year old seedlings grafted onto M26 rootstocks and blight severity was scored six weeks after inoculation.  Based on the results, 5% of genotypes were rated as resistant, 17% moderately resistant, 18% moderately susceptible, 28% susceptible and 32% highly susceptible. Based on these results, it is concluded that local APPLE germplasm is relatively susceptible to fire blight.