Studies on Distribution of Sugarcane Yellow Leaf Virus in Commercially Grown Sugarcane Varieties in Andhra Pradesh

Sugarcane (Saccharium officinarum) is an important commercial crop of the world belonging to C4 family and is principal sources of sugar, ethanol and jaggery globally (Gulati et al., 2015; Misbah et al., 2017). India ranks second in sugarcane production after Brazil and first in sugar production. Sugarcane crop is attacked by more than 200 diseases caused by fungi, bacteria, viruses, phytoplasma and nematodes resulting in severe yield losses worldwide. Among the diseases of sugarcane, Sugarcane yellow leaf disease (YLD) is one of the most prevalent diseases of sugarcane worldwide. Sugarcane yellow leaf virus (SCYLV) is a distinct member of the Polerovirus genus of the Luteoviridae family. SCYLV is the major limitation to sugarcane production worldwide and presently posing a major threat to sugarcane cultivation. The disease was first reported in Hawaii in the late 1988 and was subsequently observed in almost all sugarcane growing countries (Comstock et al., 2002). In India, it was reported during 1999 (Viswanathan, 2002). Later, the disease was reported from almost all the sugarcane growing regions in India (Maharashtra, Bihar, Uttar Pradesh, Punjab, Kerala, Tamil Nadu, Madhya Pradesh, Haryana and Andhra Pradesh). Disease incidence up to 100% in commercial fields in susceptible cultivars was reported in Florida (Comstock et al., 1999), India (Viswanathan 2002), Island of Reunion (Rassaby et al., 2004) and in Thailand (Lehrer et al., 2008).
       
SCYLV is an emerging virus that has evolved by recombination between ancestors of the three genera (Luteovirus, Polerovirus, and Enamovirus) forming the family Luteoviridae. The genome of SCYLV has a positive- sense single stranded RNA containing six overlapping open reading frames (ORF 0-ORF 5) which is devoid of a poly(A) tail and three untranslated regions (UTRs) consisting of ~5.8  kb nucleotides.  In Andhra Pradesh, sugarcane is grown in an area of 1.26 lakh hectares with average production of 76.14t/ha {Source: Cooperative Sugar Vol 51(6) Feb, 2020)}. The disease was first recorded in Nizamabad area during 2004 (Bharathi and Kishan Reddy, 2007). The wide prevalence of the disease was observed during 2008-09 season in various sugarcane varieties used in All India Coordinated trails at Agricultural Research Station, Perumallapalle, Tirupati, (Andhra Pradesh). Later, the disease was noticed on other commercially grown varieties viz., 2003 V46, 86 V 96, Co 7219, 87 A 298, Co 86032 in Coastal and Southern regions of Andhra Pradesh. At present the disease is spreading at an alarming stage infecting almost all the varieties grown by the farmers in A.P. The disease has spread to number of ruling varieties like. 2003V46, Co 86032, 83V15, 87A298, 86V96, Co 62175, 2002V48, 2005T16 (Hemalatha et al., 2014). The per cent disease incidence up to 30.6% in plant crop and 51.0% in ratoon crops was recorded in most of the cultivated varieties in Andhra Pradesh (Suresh et al., 2020; Hemalatha et al., 2021). At present scenario, the disease has attained epidemic status in the country and the situation warranted management approaches to sustain sugarcane productivity. Hence establishing disease-free healthy nurseries is suggested to the sugar industries to manage the disease and the strategy has found success in different states. An assay for viruses plays an important role to prevent the disease during germplasm exchange and seed cane production. Many methods to overcome viral infection have become ineffective. Since last two decades, production of disease free seedlings through tissue culture techniques is playing a significant role in solving the problems of viral infection in plants. This technique is applied successfully to a wide range of agricultural crops and horticultural plants to eliminate the viral infection (Rupinder and Manish, 2017; Misbah et al., 2017). Studies regarding phylogenetic origin of SCYLV revealed that 10 different genetic groups have determined viz., BRA (Brazil), CHN1, CHN2, CHN3 (China), COL (Colombia), CUB (Cuba), HAW (Hawaii), IND (India), PER( Peru) and REU (Reunion). These genotypes were determined based on analysis of the genetic diversity of their genome using partial sequences and complete genomes (Elsayed et al., 2010; Gao et al., 2012, Chinnaraja et al., 2013; Lin et al., 2014). The main objective of the present study was to detect the prevalence and distribution of Sugarcane yellow leaf virus in sugarcane growing regions of Andhra Pradesh and to determine the diversity of SCYLV isolates in India and their phylogenetic origin.

During 2017-18, twenty five sugarcane leaf samples exhibiting typical midrib yellowing symptoms were collected from major sugarcane growing regions of Andhra Pradesh (Chittoor, Nellore, Vishakapatnam, Krishna, Prakasham and Vijayanagaram districts). The collected leaf samples were stored at -20°C until further processing. The details of the SCYLV isolates used in the study are given in Table 1.


 
RNA extraction and RT-PCR
 
RNA from the infected leaf samples were isolated using TRI reagent (Sigma, USA) as per the method described by Singh et al., (2011). The quality of the RNA was checked by Nanospectrophotometer. The forward primer SCYLV- F3: 5¢ -GCAGCAGAACGGAGGGAAGAAGTC-3¢ and reverse primer SCYLVR3: 5¢-TGAGTTTGGGCGTACAR GACACCG CC-3¢ designed  by Viswanathan et al., (2008) were used to amplify ~1110 bp of ORF1 and ORF2 regions of SCYLV genome. The cDNA was synthesized from total RNA of all the 25 samples separately using Revert Aid H Minus first stand cDNA synthesis kit (MBI Fermentas, USA) as per manufactures protocol.
 
Sequence analysis and phylogenetic relationship
 
cDNA from 12 positive samples were amplified with SCYLV-F3 and SCYLV-R3 by RT-PCR. The amplicon of 1110 bp from 12 positive samples were eluted using GenElute Gel Extraction Kit (Sigma, USA). The nuleotide sequence of each virus isolate was sequenced with virus specific primers for ORF1 and ORF2 regions. The nucleotide sequence data of 12 SCYLV isolates were analyzed using Bioedit and MEGA 7.0 (Kumar et al., 2016) to study the sequence identities/similarities with the other 22 SCYLV isolates available in GenBank database (Table 2) including partial sequence data of RdRp of  representative genotypes (BRA from Brazil, CUB from Cuba, PER from Peru and REU from Reunion Island). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) were shown next to the branches and phylogenetic tree was generated with Neighbour-Joining method.

The typical symptoms of the disease observed were intense yellowing of midribs on the abaxial surface, lateral spread of yellow discoloration to the leaf lamina followed by tissue necrosis from the leaf tip spreading downwards along the midrib and a bushy appearance of the top of the plant due to internode shortening in maturing plants (Fig 1). In some sugarcane cultivars, leaves show a pinkish discoloration of the midrib on the adaxial surface (Fig 2). The disease incidence was significantly high (60-70%) at later stage (6-7 months of age) of the crop.




 
Detection of SCYLV by RT-PCR
 
The expected size (1110 bp) fragment was successfully amplified with SCYLV-F3 and SCYLV-R3 primers by RT-PCR in 12 symptomatic samples collected from major sugarcane growing regions of Andhra Pradesh (Fig 3). Successful PCR detection of RdRp gene indicated that the SCYLV was wide spread among the most popular varieties grown in major sugarcane growing regions throughout India. Similar reports are available on the widespread occurrence of SCYLV in other parts of the world (Holkar et al., 2020).



Sequencing and phylogenetic analysis
 
The amplified fragments of 1110 bp with prominent intensity obtained from infected leaf samples were sequenced. The BLAST analysis of partial RdRp gene sequences obtained from 12 different cultivars showing >90% sequence identity with SCYLV genomes from NCBI database were deposited in GenBank. The sequence data of 12 isolates under study was deposited in GenBank database with accession numbers mentioned in Table 1
       
The multiple sequence analysis of the isolates under the study with 22 similar sequences of SYLCV isolates reported from India and abroad revealed 90.6-100% identities with IND, REU, BRA, CHN, HAW, CUB, COL and PER genotypes reported from other countries. Comparisions of the nucleotides (nt) and their deduced amino acid (aa) sequences of ORF 1 and 2 of the isolates under the study with similar sequences of SCYLV isolates available in GenBank database revealed 95.7-100% nucleotide identities among them and with other isolates SCYLV reported from India (Table 2). All the Indian isolates shared maximum identity with SCYLV-CUB isolate (MF622079) and SCYLV-COL isolate (MF622078) with 96.6-99.0 % while they shared  90.6–92.2% nucleotide identity with SCYLV isolates (REU, BRA and PER genotypes) from other parts of the world. The nucleotide sequence data presented here are in consistent with previous reports which include SCYLV, the causative agent of the yellow leaf in Brazil, USA, Australia, and Mauritius as a possible member Polerovirus of the family Luteoviridae (Rott et al., 2008; Scagliusi and Lockhart 2000). The sequence comparisons reported in this study contribute to a better understanding of the taxonomic status of SCYLV isolates throughout the world.
       
Comparision of deduced amino acid sequence of RdRp among the 12 SCYLV isolates under the study revealed maximum similarity ranging from 93.4%- 100 % with IND isolates while 93.4-98.3% similarity with SCYLV-CUB genotype (MF622079) and 93.0- 98.3% with SCYLV-COL genotype (MF622078).The other genotypes viz., CHN, REU, BRA, PER, HAW shared 86.1% - 90.6% amino acid similarity with the IND isolates under the study (Table 3).The SCYLV isolates from Cuba and Colombia showed closest homology with the Indian isolates. These findings suggest that the mixed populations of SCYLV isolates that exist across India, may be due to the movement of the SCYLV isolates in the country through infected propagative material. In a study that included 18 geographical locations worldwide, the BRA-PER genotype occurred in most sugarcane producing areas wherever as genotypes CUB and REU were found in four geographical locations only. Afterwards, several isolates of SCYLV were detected in Brazil, Colombia, Guadeloupe, Mauritius and Reunion Island, suggesting different virus introductions and/or different evolution histories of the virus after its introduction into a new environment (Abu Ahmad et al., 2006 b). On the basis of SCYLV genotypes identified in Brazil, Colombia, Cuba and Peru, Abu Ahmad et al.,(2006 b) suggested that they may have been introduced through infected planting material imported from elsewhere. Phylogenetic analysis revealed that the SCYLV isolates under the study clustered into two major groups. SCYLV isolates from India, Colombia and Cuba clustered into one group and all other isolates from the other countries ie., China, Brazil, Peru, Reunion, Kenya, Hawaii, USA formed into a separate cluster (Fig 4).  The 12 SCYLV isolates under study showed close relationship with Cuba isolate (MF622079) and Colombia (MF622078) isolate along with other 9 isolates from India. The 13 SCYLV isolates from other parts of the world formed into a separate cluster with independent clades: HAW, USA, MARITIUS, REU, KENYA, CHN, BRA, REU and PER.



       

Viswanathan et al., (2008) studied the phylogentic analysis of SCYLV isolates based on partial ORF 1 and 2 sequences of the virus genome. The sequence analysis suggested that the population that exists in India was significantly different from rest of the world.  It was revealed from his study that CUB genotype was predominant among four genotypes (BRA, CUB, IND and PER) found in India.It is remarkable to note that the isolates of India clustered with isolates of Cuba and Colombia and shared maximum sequence similarity up to 99.2%.  Similarly, The phylogenetic analyses of the entire genome of SCYLV described by Abu Ahmad et al., (2006a) revealed the occurrence of three genotypes of SCYLV (BRA, PER and REU) based on the geographical location where it was first detected; Brazil, Peru and Reunion, respectively. Additionally, a virus isolate from Cuba (that was partially sequenced) showed only 77-80% amino acid sequence identities in ORF1 with isolates of genotypes BRA, PER and REU, which suggest that the Cuban isolate represent a new genotype (CUB).

It is evident from the study that Sugarcane yellow leaf disease is threatening the sugarcane cultivation affecting almost all the varieties grown in India and abroad. The phylogenetic analysis revealed that the SCYLV isolates reported from India shared maximum nucleotide and amino acid similarity with the SCYLV-Cuban and Colombia isolates. The diversity among the SCYLV isolates used in the study showed a very less variation between them while the variability was greater with the BRA, CHN, REU, PER, HAW isolates.

The authors are grateful to the authorities of Acharya N. G. Ranga Agricultural University, Guntur, Institute of Frontier Technology, Regional Agricultural Research Station, Tirupati and Agricultural Research Station, Perumallapalle for providing necessary facilities and support to carry out the work.