Nested Real-Time PCR Assessment of Vertical Transmission of Sandalwood Spike Phytoplasma (‘Ca. Phytoplasma asteris’)

SIMPLE SUMMARY: The Sandalwood Spike disease (SSD) related to ‘Ca. Phytoplasma asteris’ has almost wiped out the sandalwood population from the forests of southern India. It is known that sap-sucking insect vectors transmit phytoplasmas; however, their transmission through seeds needs thorough investigation. We found that 38.66% and 23.23% of one-month and four-month-old seedlings, respectively, tested positive for SSD phytoplasma screened using modified real-time qPCR assays in insect-free environments. Considering the current efforts to reestablish the healthy sandalwood population and its commercial importance, these findings are worrisome. The role of some other microbes in the high mortality rates of sandalwood seedlings remains unknown and requires further investigation. ABSTRACT: The Sandalwood Spike disease (SSD)-related to ‘Ca. Phytoplasma asteris’ has threatened the existence of sandalwood in India. The epidemiology of SSD is still poorly understood despite the efforts to understand the involvement of insect vectors in SSD transmission and alternate plant hosts over the last two decades. Apart from the transmission of SSD phytoplasma through insect vectors, the information on vertical transmission is entirely unknown. Over 200 seeds from SSD-affected trees and over 500 seedlings generated using commercially purchased seeds were screened for the presence of SSD phytoplasma to understand the vertical transmission in an insect-free environment. The end-point nested PCR and real-time nested PCR-based screening revealed an alarming rate of 38.66% and 23.23% phytoplasma positivity in one-month and four-month-old seedlings, respectively. These results were further validated by visualizing the phytoplasma bodies in sandalwood tissues using scanning electron microscopy. The presence of phytoplasma DNA in the seeds and seedlings is a concern for the commercial distribution of sandalwood seedlings in the current setup. This also poses a fear of spreading the disease to newer areas and negatively affecting the economy. The seedling mortality was also suspected to be associated with isolated bacterial and fungal isolates such as Erwinia, Curtobacterium, Pseudomonas, Rhodococcus, Aspergillus, Fusarium, and Neofusicoccum isolated using a culture-dependent approach. These findings strongly recommend the accreditation of commercial production of sandalwood seedlings curtailing SSD phytoplasma’s menace. Additionally, a new nested end-point and qRT PCR assays developed in this study proved valuable for the rapid screening of phytoplasma in many plant samples to detect phytoplasmas.