شناسایی مولکولی برخی از عوامل ویروسی و ویروئیدی در درختان مرکبات با علائم زوال در استان مازندران

In recent years, citrus decline disorder has significantly spread in the central areas of Mazandaran province. Affected trees exhibit symptoms such as dieback, paleness, reduced growth, stem pitting, lack of germination, weakness, gummy bark, and, in some cases, scaly bark accompanied by rapid decline. In this research, citrus tristeza virus (CTV), citrus psorosis virus (CPV), citrus exocortis viroid (CEVd) and hop stunt viroid (HSVd) were investigated in citrus trees that display these decline symptoms. For this purpose, during Autumn 2023, and Winter and Spring 2024, trees showing decline symptoms were sampled from the central regions of Mazandaran province including Sari, QaemShahr, Simorgh, Juybar and Babol. A total of 76 samples were randomly collected, representing trees with an average age ranging from 6 to 40 years and including cultivars such as Thomson orange, Blood orange, local orange, Citrus Unshiu, Page Mandarin, and rootstocks like sour orange, citrange, and citrumelo. Total RNA was extracted from all samples taken from the vein, and petiole. Subsequently, all samples were tested by reverse transcription-polymerase chain reaction (RT-PCR) using specific primers to amplify a segment of the coat protein (CP) gene of citrus tristeza virus (CTV-CPF/R) and citrus psorosis virus (CPV1-F/CPV2-R), as well as specific primers for citrus exocortis viroid (CEVd-F1/R1) and hop stunt viroid (HSVd-F1/R1). Results of agarose gel electrophoresis revealed that the PCR assay successfully amplified a fragment of approximately 504 bp in size from the CP gene of CTV in 36 samples, while no DNA fragment of the expected size was amplified for CPV. The evaluation of PCR products indicated the amplification of a 371 bp fragment for CEVd (in 46 samples) and a 299 bp fragment for HSVd (in 48 samples). Among the samples infected with CTV, 34 samples had sour orange rootstock, one had citrumelo rootstock, and one had citrange rootstock. In samples infected with HSVd, 42 samples had orange rootstock, four samples had citrumelo rootstock, and two samples had citrange rootstock. In the CEVd infected samples, 39 samples had orange rootstock, four samples had citrumelo rootstock, and three samples had citrange rootstock. To validate the PCR results, five isolates of CTV (one from each region), five isolates of CEVd, and five isolates of HSVd were sequenced in both directions. BlastN search showed that the sequenced CTV isolates shared 97.50 to 99.80% nucleotide identities with other isolates recorded in GenBank. While the sequenced isolates of HSVd and CEVd showed high nucleotide diversity and shared 92 to 100% and 93.80 to 99.50% identities with their counterpart isolates available in GenBank, respectively. Results of this research provide a report on the status of some viruses and viroids associated with citrus decline disorder in the affected areas in Mazandaran province during the current year.