Introduction

Sugarcane mosaic caused Sugarcane mosaic virus (SCMV) and Sorghum mosaic virus (SrMV) is an important viral disease that occurs widely in major sugarcane growing locations around the world. Sugarcane streak mosaic caused by Sugarcane streak mosaic virus (SCSMV) is another important viral disease that has so far only been found in Asia. In China, especially in Yunnan and Guangxi Provinces, investigations showed that incidence of mosaic/streak mosaic were generally higher than 30% and reached up to 100% in susceptible cultivars at the time of an outbreak, The high incidence of these diseases resulted in low seed cane germination (10.6–35.3%), and losses in cane yield (3–50%) and sucrose content (6–14%) (Huang and Li 2011; Huang et al. 2007). This leads to annual economic losses of hundreds of millions of Chinese Yuan, severely restricting the sustainability and stability of the sugar industry in China. The viruses causing mosaic symptoms in sugarcane are complex, consisting of several different virus species and numerous strains (Rott et al. 2008). At present, SCMV, SrMV and SCSMV are known to infect sugarcane under natural infection conditions (Shukla et al. 1992; Seifers et al. 2000; Chatenet et al. 2005). Three other viruses—Maize dwarf mosaic virus (MDMV), Johnsongrass mosaic virus (JGMV) and Zea mosaic virus (ZeMV) causing mosaic symptoms in sugarcane only after artificial inoculation (Jiang et al. 2009).

Yunnan is the second largest cane-growing area in China, with the highest potential for continued future development. However, due to the lack of comprehensive investigations on virus incidence and pathogen detection, especially in recent years, sugarcane varieties were improved and updated faster, and they were frequently allocated among the cane regions, making virus was spread to all over the sugarcane planting area through exchanged seed cane. Additionally, because of longer growth period, long continuous cropping, stubble cultivation, vegetative propagation and diversified planting system of sugarcane, mosaic/streak mosaic has become a very common and serious main disease in Yunnan. Moreover, the distribution, occurrence and damage caused by SMD and infection rates in new varieties remain unclear, making effective control of sugarcane mosaic/streak mosaic disease difficult. In this study, we sampled sugarcane fields and surveyed the occurrence of mosaic/streak mosaic in new varieties from national regional trials in Kaiyuan, Mile and Yuanjiang, Yunnan Province, in June 2015. A reverse transcription-polymerase chain reaction (RT-PCR) technique was used to detect the pathogen using nucleotide sequences of the coat protein (CP) gene. Phylogenetic analysis of genetic diversity, group composition and geographic correlations were then carried out with the aim of providing a scientific basis for targeted breeding of resistant varieties. The findings provide a background for further development of sugarcane production and effective control of SMD.

Materials and methods

Samples

Seventy-seven mosaic leaf samples of new sugarcane varieties from national regional trials in Kaiyuan, Mile and Yuanjiang, Yunnan, China, were collected in June 2015 (Table 1). The samples were sealed in plastic bags and stored at −80 °C until use. For each virus detection method, RNA of a positive control and known viral species was provided by Yunnan Key Laboratory of Sugarcane Genetic Improvement, Kaiyuan, China. RNA of a virus-free healthy sugarcane plant obtained through meristem-tip culture was used as a negative control and double distilled water was used as a blank control.

Table 1 Virus detection in new sugarcane varieties showing mosaic symptoms in China
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RT-PCR detection of SCSMV, SrMV and SCMV

The specific primers of SCSMV, SrMV and SCMV (Table 2) were designed as described by Jiang et al. (2009) and Li et al. (2011), and synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). Total RNA was extracted from leaf tissue (0.2 g) of each mosaic sample using the TransZol Plant Genomic RNA Kit (TransGen Biotech Co., Ltd., Beijing, China) according to the manufacturer’s protocol. RNA pellets was resuspended in 30 μL RNA free water, and 5.0 μL samples analyzed by electrophoresis on a 1.0% agarose gel to determine the integrity of the total RNA. The quality of the extracted RNA was assessed using an AG22331 protein and nucleic acid analyzer (Eppendorf, Germany). RNA extracted from the leaf samples was used as a template to synthesize first strand cDNA using oligodT(18) and the TransScript One-Step gDNA Removal and cDNA synthesis SuperMix (TransGen Biotech Co., Ltd., Beijing, China) according to the manufacturer’s instructions.

Table 2 Primers used to detect three virus species in sugarcane varieties showing mosaic symptoms in China
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Virus-specific primers of SCSMV, SrMV and SCMV were used for reverse transcription and PCR amplification respectively. PCR amplification of SCSMV was carried out in a 20 μL reaction mixture containing 9.6 μL ddH2O, 8.0 μL 2 × Easy Taq PCR SuperMix (TransGen Biotech Co., Ltd.), 2.0-μL of cDNA template and 0.2 μL of each primer (20 μg/μL). The amplification programme protocol was as follows: 5 min at 94 °C followed by 35 cycles of 30 s at 94 °C, 30 s at 55 °C and 1 min at 72 °C, with a final extension of 10 min at 72 °C. PCR amplification of SCMV and SrMV was carried out in a 20 μL reaction mixture containing 7.2 μL ddH2O, 10.0 μL 2 × Easy Taq PCR SuperMix (TransGen Biotech Co., Ltd.), 2.0 μL of cDNA template and 0.4 μL of each primer (20 μg/μL). The amplification programme was as follows: 5 min at 94 °C followed by 35 cycles of 30 s at 94 °C, 30 s at 60 °C and 1 min at 72 °C, with a final extension of 10 min at 72 °C. PCR products were analyzed by electrophoresis on a 1.5% agarose gel.

Cloning and sequencing of the RT-PCR products

The RT-PCR products were excised from the gel and purified using the TIANgel Midi Purification kit (TIANGEN, Beijing, China). DNA fragments were ligated into plasmid vector pMD18-T (Takara, Dalian, China), and the recombinant plasmids introduced into Escherichia coli strain DH5ɑ (Sangon, Shanghai, China) by transformation. Six positive clones from each sample were selected and the inserts sequenced at BGI Sequencing Co. Ltd. (Beijing, China).

Sequence and phylogenetic analysis

Sequencing results were analyzed with homology alignment using a BLAST search against the NCBI (http://www.ncbi.nlm.nih.gov) sequence database, and compared with published sequences in GenBank. Multiple sequence alignment was performed with DNAMAN, version 6.0 (Lynnon Biosoft, U.S. A). Based on the alignment results, CP gene sequences of SCMV, SrMV, and SCSMV were downloaded from GenBank for phylogenetic analysis. Phylogenetic trees were then generated by the maximum-likelihood method with 1000 bootstrap replications using the Molecular Evolutionary Genetics Analysis (MEGA) software program, version 6.0 (Tamura et al. 2013).

Results

RT-PCR detection of SCSMV, SrMV and SCMV

Three viruses, SCSMV, SrMV and SCMV, were found in the 77 samples (Table 1). SCSMV was found in all samples (100%). Twenty-one samples (27.2%) were infected with SrMV, while only one sample (Qiantang 5) (1.3%) was infected with SCMV. A total of 20 of 72 new sugarcane varieties were found to be infected with both SCSMV and SrMV, while Qiantang 5 was co-infected with all three virus species. The remaining samples were infected with SCSMV only.

Sequence and phylogenetic analysis

Nucleotide sequences of the CP gene of 48 SCSMV positive isolates were subsequently cloned and sequenced. Sequence identitits ranged from 97.7 to 100% (GenBank accession numbers: KX420953-KX421000). All SCSMV CP nucleotide sequences available in GenBank were also downloaded to construct a maximum-likelihood tree with the sequences obtained in this study, using the CP nucleotide sequences of Triticum mosaic virus (TriMV) (GenBank accession number: NC012779) (Fellers et al. 2009) as an outgroup. The phylogenetic tree revealed an obvious geographic difference for SCSMV, with all SCSMV isolates from China clustered into the China group. Moreover, isolates from different new varieties were distributed across all subgroups or clustered into independent branches within the China group (Fig. 1). Then, isolates from other countries were located outside the China group with an exception of an India isolate (GenBank accession number: DQ866749).

Fig. 1
figure 1

Phylogenetic tree reconstructed with CP nucleotide sequences of SCSMV. Numbers at each node are the bootstrap values of the maximum-likelihood tree (1000 replications). The homologous sequence of a Triticum mosaic virus (TriMV) isolate was used as the outgroup

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Nucleotide sequences of the CP gene of 17 SrMV positive isolates were also cloned and sequenced, revealing sequence identity of 89.0 to 99.7% (GenBank accession numbers: KX396438-KX396452, KX396453-KX396454). All SrMV CP nucleotide sequences available in GenBank were used to construct a maximum-likelihood tree with the CP nucleotide sequences of SCMV (GenBank accession number: NC003398) (Chen et al. 2002) as an outgroup. In this tree, all SrMV isolates were clustered into two groups, with different subgroups in each. The 17 SrMV isolates from the new sugarcane varieties in China were distributed among the different groups and subgroups with no obvious association with the geographical origin of the virus isolates (Fig. 2).

Fig. 2
figure 2

Phylogenetic tree reconstructed with CP nucleotide sequences of SrMV. Numbers at each node are the bootstrap values of the maximum-likelihood tree (1000 replications). The homologous sequences of an isolate of Sugarcane mosaic virus (SCMV) was used as outgroup

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Discussion

Mosaic symptoms in sugarcane can be caused by several virus species, including SCSMV, SrMV and SCMV. SrMV and SCMV are the main species occurring in Africa, America and Australia (Yang and Mirkov 1997; Alegria et al. 2003), while SCSMV is a new pathogen that has been reported in Asia: India, Sri Lanka, Thailand, Vietnam and Indonesia (Chatenet et al. 2005; Viswanathan et al. 2008; Xu et al. 2010; Damayanti and Putra 2011; Parameswari et al. 2013; Putra et al. 2013). Historically, in China, SrMV was the main causal agent of mosaic in all major sugarcane planting areas including Fujian, Guangxi, Yunnan, Guangdon and Hainan (Lu et al. 1984; Chen 1999; Chen and Chen 2002; He and Li 2006; Li et al. 2006, 2007; Zhou et al. 2006; Wu et al. 2007; Xiong et al. 2011), while SCMV has been observed sporadically in the above areas as well as Jiangxi and Zhejiang (Zhou et al. 2006; Jiang et al. 2009). SCSMV, the causal agent of streak mosaic was first reported in China in Yunnan province in 2011 (Li et al. 2011), Because of the rapid spread of SCSMV and its high pathogenicity, is now observed frequently in Kaiyuan, Yuanjiang, Xinping and Honghe, Yunnan Province, with incidences that are higher than those of SrMV. All new sugarcane varieties tested in this study were found to be infected with SCSMV, suggesting that SCSMV has replaced SrMV as the predominant virus species causing mosaic symptoms in Yunnan. It is worth noting that Yunzhe 06–80 and Yuetang 96–86 showed asymptomatic were also infected with SCSMV in this study. According to the study by Xu et al. (2008a), 54.0 and 6.0% of asymptomatic hybrids and 31.3 and 6.3% of the asymptomatic noble sugarcanes were infected with SCMV and SrMV, respectively. It is suggests that in order to ascertain the accurate and real occurrence of mosaic/streak mosaic disease, a more wide ranging sampling regime will be taken in future survey.

Simultaneous infections of sugarcane with two or more viruses differ around the world (Chen and Chen 2002; Rao et al. 2006; Viswanathan et al. 2009). However, most sugarcane samples showing mosaic symptoms tend to be infected with only one virus species, and mixed infection probability is relatively low (Xie et al. 2009). Nevertheless, in our study, co-infection with two viruses, SCSMV and SrMV, was observed in 20 new varieties (26%). Furthermore, co-infection with all three viruses (SCSMV, SrMV and SCMV) was observed for the first time in the new variety Qiantang 5.

In the SCSMV phylogenetic tree, all Chinese SCSMV isolates were clustered into the China group and those from different countries in a different group in this study. This suggests that SCSMV has obvious geographic specificity. It is different from Viswanathan et al. (2008) reported that SCSMV isolates from India, Australia, South Africa and USA were distributed in 14 phylogenetic groups and the grouping pattern revealed that the virus isolates could not be grouped based on geographical origin of the host varieties or longevity of the host variety. Interestingly, an India SCSMV isolate (GenBank accession number: DQ866749) were located inside the China group, this result suggests a common geographical origin and/or a common ancestor strain of the China isolates and this Indian isolate. For India SCSMV isolates, they were distributed in each group, suggesting higher genetic diversity and possibly different population types.

Phylogenetic analysis of the SrMV CP nucleotide sequences revealed that isolates from the new varieties in this study were distributed across the entire phylogenetic tree, clustered into different groups and subgroups with no obvious geographic differences. However, Xu et al. (2008b) reported that the phylogenetic analysis of CP nucleotide sequences of 28 SrMV isolates, including 10 SrMV from southern China, were separated into two groups associated with the hosted plants. Zhang et al. (2015) reported 66 SrMV isolates obtained from Guangxi, Yunnan, Hainan and Guangdong in China that were classified into three subgroups: I, II, and III with a 95% similarity level, and suggests that the virus have host preferences and tend to be more prevalent in certain provinces. Subsequently, the phylogenetic analysis of 113 Chinese isolates and 2 Burmese isolates and 73 isolates reported worldwide revealed that all of the SrMV isolates were clustered into three major lineages encompassing six phylogroups/genotypes. Moreover, SrMV-G5 was identified to be new distinct phylogroup that was restricted to the Fujian and Guangxi provinces, and the unique SrMV-G6 phylogroup only occurred in Yunnan province (Luo et al. 2016). Although the SrMV phylogenetic tree of this study showed the important genetic diversity occurs within SrMV in China, the similar situations were occurred in other countries (Grisham 1994; Grisham and Pan 2007). This diversity most likely indicated that the virus has been present for quite some time in China and that the virus easily adapted to changes of local conditions (appearance of several genetic variants).

Incidence and distribution of viruses causing mosaic symptoms are closely related to the resistance level of each sugarcane cultivars. Planting of susceptible cultivars in large areas plays a key role in the epidemiology of mosaic/streak mosaic. Screening, breeding and planting of resistant cultivars is therefore the most economical and effective measure of control of these diseases. However, in China, research on the identification and evaluation of sugarcane germplasm and cultivars for mosaic resistance has focused solely on SrMV (Li et al. 2013), with studies aimed at the identification and evaluation of SCSMV resistance yet to be reported. Efforts should therefore be made to adjust the strategy of sugarcane breeding using streak mosaic resistant cultivars by increasing screening of sugarcane sources resistant to SCSMV. Accordingly, a scientific basis and resistant source for selective breeding is required if mosaic and streak mosaic diseases in sugarcane are to be effectively controlled.