1. Introduction
Bacterial blight (BB) caused by
Xanthomonas oryzae pv.
oryzae (
Xoo) is amongst the oldest known bacterial diseases in Asia [
1,
2]. It is the most serious bacterial disease in many rice growing regions of the world [
3]. The
Xoo strain enters through hydathodes, stomata and wounds on the roots or leaves which causes leaf wilting, affects photosynthesis that results in yield loss and can reduce rice yield by as much as 20–80% [
4]. BB causes serious loss of rice production in Asia, Australia, Latin America, Africa and the United States [
5,
6,
7]. It is particularly destructive in the rice growing tracts of Asia during monsoon season. At the seedling stage under high atmospheric temperatures (28–34 °C) sometimes
Xoo infection causes the death of the central shoot, leading to complete crop loss [
1,
8].
Xoo isolates collected from and across Asia, Africa, and Australia exhibit high genetic diversity based on the polymorphism of transposable elements, a-virulence genes, insertion sequences, rep/box elements and other markers [
1,
9]. Based on the virulence of
Xoo strains in particular host genotypes, several distinct races have been identified [
1,
10]. Around 30 races of
Xoo have been reported globally [
1,
11]. Studies on
Xoo pathotype diversity revealed 6–11 pathogenic races based on their virulence to
Xa/xa differential lines only in India [
1,
11,
12]. Among the total known resistance genes,
Xa4,
xa5,
Xa7,
xa8,
Xa11,
xa13 and
Xa21 should be targeted as important candidates for resistance breeding against BB races in southwestern Asia.
There are different conventional control measures of BB such as antibiotics and application of copper compounds. The increasing trend of rice monoculture has spurred the development and emergence of new and more virulent races of Xoo, causing ineffectiveness of most of the chemical means of disease control. However, the development of resistant cultivars by incorporating major resistance (R) gene(s) has been proved to be the most effective, economical and eco-friendly strategy to control BB.
A total of 47 R genes (
Xa1 to
Xa47) have been identified [
1,
13]. Out of these, 14 are recessive genes, while some display semi-dominance (e.g.,
Xa27). Fourteen of the total R genes such as
Xa1,
Xa3/Xa26,
xa5,
xa13,
Xa10,
Xa21,
Xa23,
xa25, and
Xa27 have been cloned and characterized indicating the involvement of multiple mechanisms of R-gene-mediated
Xoo resistance [
2,
14]. The majority of the R genes have been tagged with closely linked molecular markers and are being used in marker-assisted selection for gene pyramiding [
1,
2,
14]. Some genes, e.g.,
Xa21,
Xa22,
Xa23,
Xa3/Xa26,
Xa31(
t) and
Xa39 confer resistance to a broad spectrum of
Xoo races, whereas others are effective against a limited number of localized BB races.
Xoo race-specific resistance in rice is controlled by both major R genes with qualitative effect, and by quantitative trait loci (QTL) that condition for partial resistance [
1,
15]. The R genes could possibly be defeated to lose their qualitative nature and express intermediate phenotypes [
1,
16].
The identification of sources of novel genetic loci regulating host plant resistance is crucial to develop an efficient strategy followed by screening, mapping, cloning and breeding. The search for a novel source of resistance is a continuous process, as the breakdown of resistance occurs due to the appearance of virulent
Xoo races [
5,
17]. With ever-evolving pathogens and changing climate patterns, it is now essential to know the status of the resistance gene(s), to expand genetic resources with novel BB resistance genes, and to deploy and pyramid them in breeding programs for durable resistance to
Xoo. Identification and isolation of novel host resistance and pathogen a-virulence genes are required for a broader understanding of mechanisms involved in host–pathogen interactions and also to determine the resistance breeding approaches. The wild species often contain untapped resources of distinct alleles useful for breeding programs. For this purpose, normally tightly linked molecular markers are exploited in order to identify genotypes with multiple resistance genes. Molecular markers offer an opportunity to characterize the germplasm collections for the existence of various resistance genes. Several markers specific to BB resistance genes have been previously studied [
5,
17,
18,
19,
20]. The marker aided selection (MAS) approach has proved its efficiency in breeding programs to improve rice genotypes against disease which allows the introgression/pyramiding of single/multiple resistance genes in a genotype with desirable traits [
5,
6,
17,
21].
Wild ancestors of cultivated rice, a natural genetic resource contain a large number of excellent genes. Along with ordinary wild rice (
Oryza rufipogon Griff.), the granular wild rice (
O. meyeriana Baill.) and the pharmaceutical/medicinal wild rice (
O. officinalis Wall.) are also well-known genetic resources. Different species are categorized into 10 genome types, six are diploid (AA, BB, CC, EE, FF, and GG) (2n = 2x = 24) and the other four are allotetraploid (BBCC, CCDD, HHJJ, and HHKK) (2n = 4x = 28) [
22]. Medicinal wild rice (
O. officinalis) belongs to the CC genome. The
O. officinalis genome is 1.6 times larger than the AA genome of cultivated
O. sativa, mostly due to proliferation of Gypsy type long-terminal repeat transposable elements, but overall syntenic relationships are maintained with other
Oryza genomes (A, B, and F) [
17]. With its diverse ecology, it has been distributed in the Yunnan, Guangdong, Guangxi and Hainan provinces of China. Its higher genetic diversity has resulted in higher resistance and tolerance to many diseases including BB.
In this study, the O. officinalis genotypes from the Yunnan province of China were crossed with O. sativa subsp. indica HY-8 and their hybrids were screened for BB resistance genes deployed through natural selection and molecular selection in wild rice germplasm. The wild rice demonstrated BB resistance in the absence of major known BB-resistance genes. The exploration of BB in its descendants provides a theoretical basis and data support. This information will aid in the further utilization of the wild rice germplasm, and in deciding gene pyramiding programs for BB resistance genes in high yielding rice varieties.
4. Discussion
Rice is an important crop contributing to global food security and grows in almost all ecosystems [
27]. Rice production is being affected by various biotic and abiotic stresses. Among these stresses, BB caused by
Xoo results in a significant reduction in global rice yield. It particularly has devastating effects in Asian countries including China, Pakistan and India [
28]. Genetic diversity is always required for any successful rice breeding program [
28]. Historically, BB has occurred epidemically and is now found in almost all major rice growing areas of Asia [
27]. This study aimed to reveal the novel source of BB resistance in rice. Hence, a medicinal rice plant (
O. officinalis) was evaluated and found as resistant even in the absence of historically known resistance (R) genes for BB.
Germplasm screening may lead to the identification of both narrow and broad sense resistance to various types of bacterial blast including leaf and neck blast [
29,
30]. In various studies, a geographically diverse mixture of blast isolates has been used to identify the stable QTL/gene(s) [
31]. To date, forty-seven genes have been identified that induce resistance against broad spectrum or race specific resistance to
Xoo [
27]. Evolving environmental conditions could cause the emergence of new pathogenic variants. Hence, a fresh effort to reveal new sources of resistance in wild material may be helpful to generate longer-term resistance to BB in cultivated species.
The current study was conducted to screen the medicinal wild rice plants O. officinalis and its hybrids to characterize against BB. The evaluation of medicinal wild parent plants and their F1, F2 and backcross individuals for BB resistance by traits, specific morphological and gene specific molecular markers revealed novel sources of BB resistance. It further highlighted the chromosomal substitutions in genomic and promoter regions of inbred and recombinants with a reference to their parental wild genotypes.
The molecular marker survey revealed that
O. officinalis does not contain the evaluated 20 reference genes. The markers-based amplification of genomic segments indicated the absence of a few targeted R genes segments in hybrid plant HY-8 but they were available in a few of the other progenies. Twenty-four primer pairs which were not amplified in
O. officinalis and detected in progenies indicate the absence of targeted segments in
O. officinalis (
Figure 3F,G). Hence, it may be an indication of an unknown source of resistance in
O. officinalis. Similar results were observed in a few recent studies, in which they used a molecular survey to screen the potentially resistant landraces and the tested genes were not found [
28]. The identification of new genes and manipulation of
O. officinalis in rice breeding may increase BB resistance since most of the genes are losing their durability and effectiveness [
32].
The sharing of common R-gene segments or the availability of homolog genomic regions indicated a common pedigree. The sources of two genomes may have common parents with HY-8, and the other genotype as
O. officinalis. Nonetheless, the absence of the downstream sequences of
Xa3/Xa26 gene coding region in
O. officinalis indicated the availability of alternate and unrevealed causes of BB resistance. The sequencing analysis revealed the genetic differences between F
2-3 from
O. officinalis and F
2-4 (
Figure 4A). Hence, it illustrated that the two parental genotypes had a new type of mutation. We also observed that
O. officinalis, HY-8 and their descendants did not contain the
Xa3/Xa26 gene but may contain
Xa3/Xa26 homologous genes.
The sequence similarity of genomic and promoter regions for
Xa4 and
Xa23 genes among HY-8, F
2-3, FD-3, F
2-4, No. 10 and the donor sequence in the resistant cultivar IR64, indicated the availability of these genes in HY-8 and its hybrids. It was observed that the five studied materials had exactly the same protein as in
O. rufipogon, but their promoter regions were missing the 38 bp region, which created a resemblance to the susceptible
xa23 gene and allelic differences in the
Xa23 functional region (EBE avrXa23) [
2,
33]. Hence the recombinant progeny HY-8 contains a susceptible
xa23 and not the disease-resistant gene
Xa23. Similarly, the resemblance of the promoter region of
Xa27 sequence as the
O. minuta and observed variation in protein coding regions resulted in inconsistent resistance in the progeny. A previous study has also been reported for the promoter region variation of the
Xa27 [
2,
34]. The similarity with two differences in genomic region sequence of
Xa27 in susceptible cultivar IR24 and small grain wild has been reported. The promoter region of the susceptible genotype had an insertion of 10 bp at about 1.4 kb upstream of the ATG and an insertion of 25 bp before the TA frame. It may not only cause the variation in the promoter region but also affect the gene function from resistant
Xa27 to susceptible
xa27. Furthermore, the similarity in the promoter region of
Xa27 with
O. officinalis in F
2-3 and F
2-4 and a variation in the coding region was observed. Hence, we propose that
O. officinalis, HY-8 and the progeny only possessed the homolog of
Xa27, while the F
2 generation may also contain
Xa27 resistance or susceptible allele
xa27.
The wild species are valuable sources of potential genes for tolerance or resistance to abiotic and biotic stresses and are helpful for revealing the gaps in genetic diversity [
28,
35]. In the case of bacterial blight, many important genes such as
xa5,
xa13 and
Xa21 have been identified from cultivated rice and wild species [
2,
36]. Previously, a dominant gene
Xa21 was identified from a wild rice parent
O. longistaminanta, showed resistance to all six races of BB in Philippines [
27]. However, it was defeated and broken down in other Asian countries such as Nepal, Thailand and India [
27]. Another similar study was conducted on wild rice
O. malampuzhaensis and
O. rufipogon. They reported the susceptibility of
O. malampuzhaensis for all 20 tested
Xoo strains in the absence of
Xa21 but
O. rufipogon showed resistance without
Xa21, which may due to the availability of a new major gene.
O. rufipogon was also identified as source of resistance to BB in China [
27,
37] and found the major BB resistance gene
Xa23 [
37]. Other similar studies have been reported for the presence of BB resistance in
O. minuta Presl. [
38] and
O. latifolia [
39].
The current study revealed that in addition to carrying the Xa1, Xa2, Xa3/Xa26, Xa14, Xa23, Xa31(t) and Xa45(t)4 homologous genes, the hybrid of O. officinalis also contained the parts from the xa27 susceptible region and had lesser parts from resistant Xa27, which may have come from the medicinal wild parent and directly contributed to BB resistance. The genotypes without the R genes exhibited a resistant response to Xoo and may be a valuable genetic resource for rice breeding for BB resistance for higher yield. The present study provides a reference for investigating medicinal rice for R gene(s) identification against BB using a forward genomics tool. The gene(s) linked to the molecular markers used for R gene assays can be used as a tool to validate the bi-parental or diverse mapping population. A genome-wide association analysis of BB resistance will help in the identification of DNA-markers.