Document Type : Research Article
Authors
Department of Plant Pathology, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran
Abstract
Introduction
Almond (Prunus amygdalus Batsch.) is a plant native to Iran, and Iran is one of the most important almonds producing countries in the world. Phytoplasmas are prokaryotic cells that lack a cell wall and are restricted to the phloem of their host plants and are mainly transmitted by leaf hoppers. One of the most important diseases of the almond tree is witches' broom disease, which is caused by phytoplasmas. This disease has caused great damage to almond trees in Iran and Lebanon, leading to the death of more than a hundred thousand almond trees. C.Phytoplasma phoenicium, which belongs to the pigeon pea witches' broom phytoplasma group, has been reported as the causative agent of almond witches’ broom disease. Almond witches' broom has been reported from almonds, peaches, nectarines, bitter almonds and apricots in the Iranian provinces of Chaharmahal and Bakhtiari, Isfahan, Kerman, Fars and Kurdistan. Symptoms of the disease are yellows, small leaves, witches’ broom and dieback. Almond witches’ broom disease has been reported from Khorasan Razavi province, but different symptoms were observed on almond trees in different locations of this province. In this study, almond tree orchards in Khorasan Razavi province were examined for the symptoms of almond witches’ broom and etiology of the disease was determined.
Materials and Methods
In 2022, the samples were collected from almond orchards of Khorasan Razavi province and grafted onto healthy almond seedlings and periwinkle plants. The DNA extraction from the main vein or bark of the stems was performed using CTAB method. Nested polymerase chain reaction was performed with universal phytoplasma primers (P1/P7 followed by R16F2n/R16R2), and the PCR products were electrophoresed and sequenced. After blasting in the NCBI database, phylogenetic trees were constructed using the maximum likelihood method in MegaX software with 1000 replicates in the bootstrap test.
Results and Discussion
In the northern, central and southern regions of Khorasan Razavi province, random sampling was carried out in almond plantations. In the northern region, symptoms of almond witches’ broom were observed only in Chenaran. In the central region, symptoms of witches’ broom were found in Mashhad and Nishapur. However, in the southern region, the symptoms of almond witches’ broom were more frequent and observed in Kashmar, Koohsorkh, Torbat-Heydarieh and Sabzevar. The symptoms of infected almond trees in the northern region were small leaves, mild witches’ broom and dieback. The symptoms in the central region were severe small leaves, yellows, severe stunting, witches’ broom and decline. Infested almond trees in the southern region showed severe small leaves, severe witches’ broom and proliferation, and dense growth of thin branches from the trunk. The disease symptoms in the northern areas of Khorasan Razavi were moderate and in the central and southern areas were severe. In addition, there were significant differences in the type of symptoms in the central and southern regions. In the nested polymerase chain reaction, a fragment of 1250 base pairs replicated from the infected samples. Sequencing and BLAST analysis revealed that the amplified fragments were similar to the phytoplasma 16S rRNA. Two months after inoculation, disease symptoms appeared on the inoculated almond seedlings. Almond seedlings inoculated with the northern strain showed symptoms such as terminal bud dieback, dieback of lateral branches and necrotic spots on the leaves. Almond seedlings inoculated with the isolate from the central areas showed symptoms such as yellows, severe witches’ broom, stunting and small leaves. The symptoms in seedlings inoculated with the strain from the southern area of Khorasan Razavi province were severe proliferation, severe stunting, small leaves and necrotic spots on the leaves. In periwinkle plants, isolates from the northern and southern regions induced mild yellows and phyllody; however, isolates from the central region induced severe yellows and phyllodes. The sequence blast results showed that the Chenaran isolate from the northern region of Khorasan Razavi had 98.34% similarity with 16S rRNA strain Ei1090 of C. Phytoplasma phoenicium. The 16S rRNA sequence of the Kashmar isolate from the southern region was most closely related to the Khafr isolate (Khafr, Iran) of almond witches' broom phytoplasma. These two phytoplasma strains belong to the 16S rRNA (16SrIX) group of pigeon pea witches' broom phytoplasmas that can cause almond witches' broom. However, Ei1090 is a mild strain and the Khafer isolate is a severe strain of C. Phytoplasma phoenicium. The similarity of the isolates from Chenaran and Kashmar with other members of the 16SrIX group ranged from 98.79-99.33 and 98.48-98.95%, respectively. The 16S rRNA sequence of the Mashhad isolate from the central region of Razavi Khorasan was 94.99% similar to the CPh strain of Clover phyllody phytoplasma, a member of the Aster yellows phytoplasma group (16SrI). The similarity of the isolate from Mashhad with other members of the 16SrI group was between 90.99-95.98%. In the phylogenetic tree, the isolates from Chenaran and Kashmar were located in the same branch together with the reference strain of the pigeon pea phytoplasma group (16SrXI group). In addition, the isolates from Chenaran and Kashmar were placed in the same branch with subgroups B and A of the 16SrXI group. In addition, the Mashhad isolate was classified in the aster yellows phytoplasma group (16SrI group) within subtype C, which is a new strain of subgroup C that causes almond witches' broom disease.
Conclusion
Almond witches’ broom is a devastating disease in almond orchards in the Khorasan Razavi Province. Symptoms include yellows, small leaves, witches’ broom, growth of many thin branches on the main trunk, decline and death of the almond tree. Symptoms of the disease were moderate in the northern regions and more severe in the central and southern regions of Khorasan Razavi province. The results indicate that different strains of C. Phytoplasma phoenicium and Clover phyllody phytoplasma are associated with almond witches’ broom disease in this province.
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