Detection and diversity of maize yellow mosaic virus infecting maize in Nigeria

Abstract

Maize yellow mosaic virus (MaYMV; genus Polerovirus; family Luteoviridae) was recently characterized from maize in China and subsequently detected in mixed infection with sugarcane mosaic virus (genus Potyvirus; family Potyviridae) in sugarcane and itch grass in Nigeria. This study was conducted to understand the status and genetic diversity of MaYMV in maize fields in the northern guinea savannah region of Nigeria. A survey was conducted in 2017 and maize (n = 90) and itch grass (n = 10) plants suspected of virus infection based on appearance of mosaic and/or yellowing symptoms were sampled in Kaduna (n = 65) and Katsina (n = 35) states. The samples were screened individually by reverse transcription polymerase chain reaction using the genus-specific primers targeting poleroviruses and potyviruses Pol-G-F and Pol-G-R primers encompassing the partial P1–P2 fusion protein and coat protein genes of poleroviruses and primer pair CI-For & CI-Rev encompassing the partial cylindrical inclusion proteins of most potyviruses. A subset of amplified DNA fragments was cloned, Sanger-sequenced, and the obtained sequences were bioinformatically analyzed along with corresponding sequences from GenBank. The ~ 1.1 Kb polerovirus fragment was detected in 32.2% (29/90) of the maize samples while all 10 itch grass samples tested negative. BLASTN analysis of sequences derived from six polerovirus samples confirmed the virus identity as MaYMV. In pairwise comparisons, the MaYMV sequences derived in this study shared 97–99% nucleotide identity with sequences of other MaYMV isolates available in the NCBI GenBank. Phylogenetic analysis revealed the segregation of global MaYMV sequences into three host-independent clusters with pattern of geographic structuring.

Introduction

Nigeria currently ranks tenth globally in maize (Zea mays L.) production and first in Africa [11]. About 90% of this production is attributed to smallholder farmers [3]. While Nigerian farmers have seen better access to seeds of improved maize varieties in recent years, the management of diseases and pest insects continue to be a weak link in the maize production value chain. Among the major disease constraints to maize production in Nigeria, those caused by viruses and fungi are major impediments to enhanced productivity due to their negative impacts. As for virus infections, maize streak disease caused by Maize streak virus (genus Mastrevirus; family Geminiviridae) and mosaic disease caused by Sugarcane mosaic virus (genus Potyvirus; family Potyviridae) have been documented in maize in Nigeria [23, 27, 29, 30]. In a recent study, we reported the occurrence of maize yellow mosaic virus (MaYMV, genus Polerovirus) in sugarcane (Saccharum spp.) and itch grass (Rottboellia cochinchinensis) in the northern guinea savannah region of Nigeria [28], a region regarded as the ‘maize belt’ of the country [12]. MaYMV was first characterized in maize from China [4] and is now accepted as a bona fide virus species in the genus Polerovirus (family Luteoviridae) [13]. The detection of MaYMV in sugarcane and itch grass samples obtained from farmer’s fields in the northern guinea savannah region of Nigeria [28] thus represented the first record of the occurrence of the virus in Africa. More recent reports have shown the widespread nature of MaYMV in many parts of the world with reports of its detection in maize in Burkina Faso [24], Ecuador [2], Brazil [7], Ethiopia [8], Kenya and Tanzania [19]. A recent report also documented the occurrence of MaYMV in Panicun miliaceum and Sorghum bicolor in South Korea [18], further expanding the host range of the virus.

The genus Polerovirus is one of the three established genera in the family Luteoviridae; the other two being Luteovirus and Enamovirus [13]. Poleroviruses are transmitted in a circulative, non-propagative manner by several species of aphids (Aphididae, Insecta), but not by mechanical means or through seeds [5, 16]. They possess a linear positive sense, monopartite RNA genome of about 5–6 Kb in length [5]. Members of the genus typically encode six open reading frames (ORF) referred to as ORF0–ORF5 and three untranslated regions (UTRs), including the 5′ UTR, 3′ UTR and a intergenic region between ORF2 and ORF3 [20, 21]. Poleroviruses have a wide host range including several cereal and vegetable crops. They typically cause yellowing symptoms on susceptible host plants and often cause major yield losses in vegetables and arable crops [17]. Following the detection of MaYMV in sugarcane and itch grass in Nigeria [28], we hypothesized that the virus is also present in maize plants in the study area. The results of this study support this hypothesis via a documentation of the occurrence of MaYMV in maize for the first time in Nigeria. Phylogenetic analysis of global MaYMV sequences, including those from Nigeria, indicate a host-independent but geography-specific pattern of clustering of MaYMV sequences with implications for disease management.

Materials and methods

Samples collection and total nucleic acid extraction

One hundred leaf samples exhibiting mosaic, yellowing and or stunted growth were collected from maize plants (Kaduna = 55, Katsina = 35) and itch grass (Kaduna = 10) from 15 farmers’ fields within the northern guinea savannah region of Nigeria. Each sample was dried under CaCl₂ at room temperature and then shipped to Texas A&M Agrilife Research and Extension Center, Weslaco, TX under USDA-APHIS-PPQ permit (P526P-14-04321). Total nucleic acid (TNA) extracts were prepared from each sample using the Spectrum™ Plant Total RNA kit (Sigma Aldrich, St. Louis, MO, USA) as per manufacturer’s protocol and used for virus diagnostic assays.

Reverse transcription polymerase chain reaction (RT-PCR)

The TNA extracts from individual samples were used as templates for cDNA synthesis with the ThermoScript™ RT-PCR System (Thermo Fisher Scientific, Waltham, MA, USA) as per manufacturer’s protocol. Approximately 400 ng/µL cDNA aliquots per sample was subjected to PCR using the universal polerovirus primers Pol-G-F/Pol-G-R [14] that targets the partial P1–P2 fusion protein and coat protein encoding regions. PCR assays were performed in final reaction volumes of 25 µL containing 5× PrimeSTAR GXL buffer (Takara Bio Inc., Mountain View, CA, USA), 20 µM forward and reverse primers, 2.5 mM dNTPs, 1.5 U PrimeSTAR GXL DNA polymerase, and 2 µL cDNA aliquot. The cDNA templates were also used to screen for the presence of potyviruses using a pair of degenerate potyvirus primer, CI-For & CI-Rev [9], capable of amplifying ~ 700 bp fragment of the cylindrical inclusion protein of potyviruses. The size specificities of the amplicons were verified by resolving 10 µL of each PCR product, along with the 1 Kb Plus DNA ladder (Thermo Fisher Scientific), on 1.5% agarose gels pre-stained with ethidium bromide. The gels were visualized under a UV-transilluminator (Kodak Gel Logic Imaging System; Eastman Kodak Company, Rochester, NY).

Cloning, sequencing, and sequence analyses

Amplicons from six representative samples (2 from each site: Makarfi & Damsite in Kaduna state and Danja in Katsina state) were first gel-eluted (Zymoclean™ Gel DNA Recovery Kit; Zymo Research, Irvine, CA, USA), then individually ligated into pCR2.1 TOPO-TA vector (Thermo Fisher Scientific) following the manufacturer’s instructions. The product of each ligation reaction was used to transform One Shot^® TOP10 chemically competent Escherichia coli cells (Thermo Fisher Scientific) following which plasmid DNA was purified from positive recombinant clones with the GenElute™ Plasmid Miniprep Kit (Sigma-Aldrich). Two independent clones were sequenced per isolate in both orientations and the within-isolate pairwise percent nucleotide (nt) identity values were generated with the BioEdit Sequence Alignment Editor version 7.2.5 [10]. A consensus sequence was derived when the two clones per isolate showed ≥ 98% nucleotide identities to exclude in vitro errors potentially introduced during RT-PCR and sequencing. Multiple alignments of nt sequences (Table 1) were performed with the program MUSCLE ([6]; http://www.ebi.ac.uk/Tools/msa/muscle/), using default settings.

Table 1.

The GenBank accession numbers of MaYMV published sequences and sequences derived in this study (taxon ID bolded) used for sequence comparison

Taxon ID	Country	Host Plant	Year collected	GenBank accession number
BF7	Burkina Faso	Maize	2016	KY711383
BF14	Burkina Faso	Maize	2016	KY711387
T2F2S5	Kenya	Maize	2014	MF425875
LETF2S1	Kenya	Maize	2014	MF425861
16/0128	Tanzania	Maize	2015	MG664794
16/0128	Tanzania	Maize	2015	MG664793
MV90	Ethiopia	Maize	2016	MF684368
MV115	Ethiopia	Maize	2016	MF684369
97_EA	Kenya	Maize	2017	MF974579
KDHTS	Nigeria	Sugarcane & Itch grassa	2015	KY684356
KDSIp	Nigeria	Sugarcane	2015	KY684357
KDW8p	Nigeria	Itch grass	2015	KY684358
KDM11	Nigeria	Maize	2017	MH790911
KDM16	Nigeria	Maize	2017	MH790912
KDM18	Nigeria	Maize	2017	MH790913
KDM21	Nigeria	Maize	2017	MH790914
KTM30	Nigeria	Maize	2017	MH790915
KTM34	Nigeria	Maize	2017	MH790916
YW2094	South Korea	Panicuma	2016	MF622081
MR2200	South Korea	Sorghuma	2016	MF622082
Yunnan 9	China	Maize	2014	KU291105
Yunnan 4	China	Maize	2013	KU291100
Yunnan 11	China	Maize	2013	KU248489
RMV2	China	Maize	2013	KT992824
Yunnan1	China	Maize	2013	KU179221
SP1	Brazil	Maize	2013	KY940544
M04	Ecuador	Maize	2016	KY052793

^aItch grass, Rottboellia cochinchinensis; Panicum, Panicum miliaceum; Sorghum, Sorghum bicolor

Phylogenetic analyses

The aligned nucleotide sequences were used in phylogenetic analysis along with representatives of corresponding global MaYMV sequences available in GenBank (Table 1). Phylogenetic analysis was performed with the MEGA7 program [15] using the substitution model that best fitted the protein dataset as described previously [1]. All three methods (maximum likelihood [ML; 22], neighbor joining [NJ; 26], and minimum evolution [ME; 25] were used with 1000 bootstrap replications. The tree topologies and relative clustering patterns of the sequences were compared for all three methods.

Results

RT-PCR

Maize plants exhibiting yellow stripes at the leaf margin or away from the midrib (Fig. 1a, c) were observed in farmers’ fields in Katsina and Kaduna states of Nigeria. In some cases, the symptomatic maize plants also appeared stunted relative to a healthy-looking plant of the same age (Fig. 1b). Approximately 32% of the 90 maize samples (28/90) collected from farmers’ fields gave the expected ~ 1100 bp product by RT-PCR with the Pol-G-F/Pol-G-R primers [14], indicating the presence of a polerovirus target in these samples. The polerovirus-positive samples were obtained from eight of 15 farmer’s fields, indicating a 53.3% prevalence across fields in the northern guinea savannah region of Nigeria. All the 10 itch grass samples tested negative for poleroviruses by RT-PCR (Table 2). RT-PCR analysis performed with the CI-For/CI-Rev primers [9] resulted in the detection of a potyvirus target in 32.2% (29/90) of the maize and 50% (5/10) of itch grass samples (Table 2). Further analysis of the results obtained from the maize samples revealed the occurrence of mixed infection of both viruses in 8.9% (8/90) of the samples, a polerovirus target alone in 23.3% (21/90), and a potyvirus target alone in 28.9% (26/90). The remaining 38.9% (35/90) of symptomatic maize samples were negative with both primer pairs suggesting that they may be infected with other viruses besides poleroviruses and potyviruses.

Fig. 1.

Virus-like symptoms observed on maize plants in Kaduna and Katsina states, Nigeria. The observed symptoms included (a) wide yellow stripes that may sometimes be accompanied with (b, c) stunted growth. Symptomatic plants tested positive by RT-PCR for maize yellow mosaic virus (MaYMV)

Table 2.

Relative proportions of single and mixed infections of Polerovirus and Potyvirus targets in the survey samples based on RT-PCR screening using genus-specific oligonucleotides

Location	Host	No. positives/total no. samples tested
		Polerovirus	Potyvirus	Mixed infection
Makarfi, Kaduna	Itch grassa	0/10	5/10	0/10
Makarfi, Kaduna	Maize	4/30	10/30	0/30
Dam site, Kaduna	Maize	9/25	7/25	3/25
Danja, Katsina	Maize	16/35	12/35	5/35

^aItch grass, Rottboellia cochinchinensis

Sequence analysis

The BLASTN analysis of six sequences derived in this study (2 each from 3 sites, Table 2) showed > 96% identity to the MaYMV sequences presented in the NCBI database, with 100% query coverage. These sequences have been deposited in GenBank with accession numbers KY684359-64 (Table 1). In pairwise comparisons, the maize-derived MaYMV nucleotide sequences obtained in this study shared > 99% identity and 99–100% identity with the MaYMV isolates reported from sugarcane and itch grass samples. The sugarcane and itch grass MaYMV sequences were both obtained from the same geographical region in Nigeria [28]. Thus, the results suggest that MaYMV isolates from the different locations and crop hosts in Nigeria are highly identical to each other. Further analysis showed that the MaYMV sequences obtained from the three host plants from Nigeria shared 96.3–99.9% nt identities with MaYMV isolates from other cereal-producing regions of the world. The BLASTN analysis of the potyvirus-specific sequences obtained from maize and itch grass confirmed them to be SCMV (data not shown).

Phylogenetic analysis

Phylogenetic analysis of global MaYMV isolates, performed with nucleotide sequences corresponding to the partial P1–P2 fusion protein and coat protein gene of the virus revealed their segregation into three distinct phylogroups, tentatively named as phylogroups I to III (Fig. 2). Phylogroup I consisted of MaYMV sequences reported from African countries, including Nigeria (n = 9: this study = 6, GenBank = 3), Burkina Faso (n = 2), Tanzania (n = 2), Kenya (n = 3), and Ethiopia (n = 2) (Fig. 2). Phylogroup II comprised of isolates from East Asia including China (n = 5) and South Korea (n = 2). Phylogroup III comprised of two isolates from South America, one each from Ecuador and Brazil (Fig. 2). Notably, the MaYMV clustering was independent of host plant origin (Fig. 2). Thus, it can be concluded that global MaYMV isolates segregated in a geography-specific but host-independent manner.

Fig. 2.

Unrooted minimum evolution phylogenetic tree (K2 + G + 1) depicting evolutionary relationships among global isolates of maize yellow mosaic virus (MaYMV) based on analysis of ~ 1100 bp nucleotide sequences encompassing the partial P1–P2 fusion protein and coat protein genes of MaYMV. The analysis was performed with the MEGA7 program [15] with 1000 bootstrap replications. MZ= maize, SC sugarcane, IGRS itch grass, SG Sorghum, PN Panicum

Discussion

Maize is cultivated intensively in the northern guinea savannah region of Nigeria along with sugarcane on the same or adjacent fields. Following the recent detection of MaYMV in sugarcane and itch grass samples from the region [28], we hypothesized an occurrence of the virus in symptomatic maize plants in farmers fields. Our survey studies using RT-PCR and sequence analysis confirmed the presence of MaYMV in several symptomatic maize samples tested in 15 maize farms in Kaduna and Katsina states. This is not surprising considering that maize is a major host of MaYMV and several maize-derived isolates of the virus have been characterized from different geographical locations around the world [2, 4, 7, 24]. The emerging body of knowledge on MaYMV thus indicate its widespread distribution, making it a potential threat to food security in countries such as Nigeria where maize is a staple for human consumption and an integral component of animal feed. Further studies are necessary to determine the economic impact of MaYMV when present alone or in mixed infection with other cereal-infecting viruses such as SCMV.

MaYMV in maize was detected in Asia, Latin America and Africa, however, the pathway for the spread of the virus between wide geographic regions is unknown at this time. The detection of geography-specific but host-independent clustering pattern of global MaYMV sequences suggests wide spread and local adaptation. It is conceivable that the virus may have spread through planting material of other species and inhabited susceptible perennial species in introduced regions. The detection of MaYMV in sugarcane and itch grass in Nigeria [28] supports this possibility. Subsequently, aphid vectors feeding on multiple hosts may have contributed to the movement of virus between reservoir perennial hosts and annual crops such as maize. Another possibility is that MaYMV could have spread through true seed, but this pathway of virus spread has not yet been established for poleroviruses. Further studies are required to evaluate MaYMV in wild hosts and weeds and the relative abilities of different aphid species to acquire and transmit MaYMV within and between different grass plants to understand the virus ecology and infection pathway to maize, to aid the development of an integrated disease management strategy.

A significant number of symptomatic maize (46.7% or 42/90) and itch grass (50% or 5/10) samples tested negative for poleroviruses [14] and potyviruses [9], indicating that these cohort of plants may harbor other viruses or virus-like agents. Future studies preferably using high-throughput sequencing are necessary to identify possible new virus species or strains of known viruses for better understanding of the virome of cereal crops in Nigeria.

From an epidemiological standpoint, the detection of MaYMV in perennial sugarcane and itch grass, a common weed within cereal production systems in Nigeria [28], suggest their potential roles as reservoir hosts of the virus. These perennial plants could serve as ‘green bridges’ for the survival of MaYMV (and other cereal-infecting viruses) between maize crops in the northern guinea savannah agroecological zone. Future studies are required to assess the economic impact of MaYMV on maize and sugarcane, and the effects of mixed infection of the virus with other cereal infecting viruses.