Nested and TaqMan® probe based quantitative PCR for the diagnosis of Ca. Phytoplasma in coconut palms

The root (wilt) disease caused by phytoplasma (Ca. Phytoplasma) is one of the major and destructive occurs in coconut gardens of Southern India. As this organism could not be cultured in vitro, the early detection in the palm is very much challenging. Hence, proper early diagnosis and inoculum assessment relay mostly on the molecular techniques namely nested and quantitative PCR (qPCR). So, the present study qPCR assay conjugated with TaqMan^® probe was developed which is a rapid, sensitive method to detect the phytoplasma. For the study, samples from different parts of infected coconut palms viz., spindle leaflets, roots and the insect vector—leaf hopper (Proutista moesta) were collected and assessed by targeting 16S rRNA gene. Further, nested PCR has been carried out using p1/p7 and fU5/rU3 primers and resulted in the amplification product size of 890 bp. From this amplified product, specifically a target of 69 bp from the 16S rRNA gene region has been detected through primers conjugated with Taqman probe in a step one instrument. The results indicated that the concentration of phytoplasma was more in spindle leaflets (8.9?×?10⁵ g of tissue) followed by roots (7.4?×?10⁵ g of tissue). Thus, a qPCR approach for detection and quantification of coconut phytoplasma was more advantageous than other PCR methods in terms of sensitivity and also reduced risk of cross contamination in the samples. Early diagnosis and quantification will pave way for the healthy coconut saplings selection and management under field conditions.

     

Root (wilt) disease of coconut palms in South Asia – an overview

Root (wilt) disease (RWD) caused by phytoplasma is one of the most devasting diseases of coconut palms. The major symptoms of the disease in leaves are wilting and drooping and flaccidity; ribbing, paling/yellowing and necrosis of leaflets are typical symptoms of foliar diseases. Unopened pale yellow leaflets of spindle leaves are more susceptible to leaf rot disease, which is caused by Exerohilum rostratum and Colletotrichum gloeosporioides. RWD is caused by phytoplasmas, the cell wall-less prokaryotes that are bounded by a “unit” membrane. In ultrathin sections, they appear as a complex multi-branched, beaded, filamentous or spheroidal pleomorphic bodies. The disease was transmitted by plant hoppers (Proutista moesta) and lace wing bug (Stephanitis typica). Phytoplasmas are generally present in the phloem sieve tubes and in the salivary glands of these insect vectors. Phytoplasmas cannot be cultured in vitro, and hence it is very difficult to identify them. Using polymerase chain reaction technique, group-specific primers have been applied to detect mixed-phytoplasma infections in a single host. RWD, is a non-lethal, debilitating disease, and hence an integrated approach for the management of this disease in coconut palms has been discussed in this study.     

Epidemiology of grey mildew and Alternaria blight of cotton

Grey (Areolate) mildew (Ramularia areola) and Alternaria blight (Alternaria macrospora) are two important fungal foliar diseases affecting cotton in India. Both the diseases are polycyclic in nature. The primary inoculum for grey mildew is through conidia or ascospores from infected debris and/or perennial cottons and the secondary spread is through primarily infected leaves. Whereas for Alternaria blight the spread is initially from seed-borne inoculum (in Gossypium herbaceum and Gossypium arboreum cottons) and/or crop debris and the secondary spread is from sporulating lesions on older leaves. Both R. areola and A. macrospora require a temperature regime of 20–30?°C with prolonged high humidity (>80%) and frequent rains for infection and disease development. However, it has been observed that cool weather coupled with prolonged dewy periods in the absence of rains has also been found conducive for the development of both the diseases. So, suitable epidemiological tools and models are required to predict the disease development, spread and to design suitable management practices.