1. Introduction
Asian ginseng (Panax ginseng M.) and American ginseng (P. quinquefolius L.) are two important medicinal herbs in the genus Panax, and they are mainly cultivated in the Northeastern region of China. Particularly, the region of Changbai Mountain has a long cultivation history of Asian ginseng. American ginseng has been planted in Northeast China since the 1980s. Notably, commercial growers commonly plant Asian ginseng and American ginseng on the same farm. More interestingly, they rotate the two species sequentially to avoid replant obstacles and fully utilize the farmland. Ginsengs are perennial plants, and their roots are usually harvested after four to six years of growth in the field. Such a long time of soil exposure makes them vulnerable to infection by soilborne pathogens.
In Northeast China, the most notorious disease of Asian ginseng is red-skin root, which is caused by a complex being consisting of 12 species of fungi [
1]. It is common that the complex contains
Fusarium spp. and
Rhexocercosporidium panacis. In addition, seven species of CLF anamorphs, such as
Ilyonectria species, take account for two-thirds of the total population. Similar to red-skin root of Asian ginseng, rusty root rot is another devastating disease and is also mostly caused by species with CLF anamorphs [
2].
For American ginseng, Cylindrocarpon root rot and rusty root rot are the most important root diseases. Although Northeast China is an important ginseng-producing region, these diseases have not been well described. Cylindrocarpon root rot of American ginseng is also called disappearing root rot due to the symptoms of complete decay of diseased roots. It is mainly caused by
Cylindrocarpon species, which have been well documented in Canada [
3,
4]. Rusty root rot of American ginseng can be caused by several
Fusarium species and
R. panacis [
5,
6]. In China, several
Fusarium species, including
F. solani,
F. oxysporum,
F. dlamini and
F. armeniacum have been reported to cause disappearing root rot in American ginseng [
7,
8].
Cylindrocarpon destructans,
C. panacis,
C. obtusisporum and
C. panacicola have been reported sporadically to cause rusty root rot [
9,
10]. Overall, fungi with CLF anamorphs are dominant pathogens causing root diseases in both Asian ginseng and American ginseng. It is not clear whether the pathogenic CLF differed in pathogenicity and virulence on both Asian ginseng and American ginseng.
It is a challenge to effectively manage such a pathogen complex in ginseng root disease control. Clarification of the population structure and host specificity of pathogens will be significant for making targeted disease management strategies. Geographic factors may affect the pathogen complex through temperature, humidity and other conditions. In addition, some CLF species not only infect
Panax species but have a broad host range from herbaceous plants to woody plants, such as
Lilium sp.,
Vitis vinifera,
Thymus sp.,
Quercus spp.,
Protea sp. and
Leucospermum spp. [
11]. Genetic diversity of
C. destructans from Asian ginseng in Korean has been associated with hosts based on RAPD finger printing or sequence analysis of nuclear ribosomal gene internal transcribed spacer (ITS) and mitochondrial small subunit (mt SSU) rDNA; however, in the study, only less than 20 isolates were investigated [
12,
13]. Although there are no genetic markers employed specifically in genetic study of CLF, several phylogenetic analyses of CLF showed that histone H3 (
his3) gene had the highest resolution among several gene loci [
1,
11].
In the present study, our aims were to (i) identify the CLF isolates causing American ginseng root rot in Northeast China; (ii) determine the cross-pathogenicity between Asian ginseng and American ginseng; (iii) characterize the genetic structure of the CLF isolates from ginseng across the main growing regions in Northeast China; and (iv) investigate the geographic and host-origin effects on the population structure. Knowledge of the population genetics of CLF isolates would be important for developing sustainable and effective strategies for ginseng root disease control.
4. Discussion
We provided a whole picture of the pathogen complex of CLF populations associated with ginseng root diseases in Northeast China by analyzing 169 isolates. Among the CLF species,
I. robusta,
I. communis and
I. mors-panacis are the top three most frequently isolated species causing ginseng root diseases in Northeast China, which was also supported by previous studies [
1,
2]. Although
I. robusta and
I. communis were more frequently isolated than
I. mors-panacis, they were less virulent than
I. mors-panacis.
Ilyonectria mors-panacis usually causes root rot on ginseng and was a highly virulent CLF species shown in this and other studies [
24,
25,
26].
Ilyonectria communis is widely distributed in Northeast China, and its host range should be addressed in future research. In addition to
Ilyonectria species causing ginseng root diseases described in the present study,
I. crassa and
I. panacis have been reported to cause root diseases in American ginseng in Canada [
11].
We have provided the first in-depth assessment of the genetic structure of CLF populations associated with ginseng in Northeast China, which were clustered into two distinct genetic groups. The two groups were discriminated against by Changbai Mountain. The Changbai populations generated more haplotypes and higher nucleotide diversity than the other populations. The high and significant pairwise FST value between two geographical groups and the genetic variation contributed by geographical regions also supported the two distinct genetic clusters. This could be due to a similar climate and longer ginseng growing history along the range of Changbai Mountain than in other areas in Northeast China. The Changbai group was even distinctly different from all other groups, including worldwide isolates. The retrieved Northeast group was close to the non-Changbai group of this study. This provides another evidence for the Changbai group with unique genetic populations.
We found a significantly positive correlation between genetic distance and geographic distance among CLF populations. This indicated that geographic and climatic factors contributed to the overall genetic differentiation in CLF populations associated with ginseng plantations. A similar result has been found in
I. liriodendri causing black foot disease in New Zealand vineyards in a previous study [
27]. Besides the local climate, the soilborne
Ilyonectria species are soilborne and limited for long-distance spread, and their reproduction is restricted locally. Although seedling transportation can cause a long-distance spread of soilborne pathogens, the current data implied this was not a main contributing factor.
There was no tight association of CLF populations with their hosts
P. ginseng and
P. quinquefolius in Northeast China, based on STRUCTURE analysis. The conclusion could have been strengthened if the genetic relationships among these host populations were examined by using discriminant analysis of principal components and Bruvo genetic distance [
28]. However, CLF isolates showed cross-pathogenicity in these two hosts, indicating their low host specificity. Interestingly, we found most CLF species had a higher virulence on
P. ginseng than on
P. quinquefolius. This partially explains why it is practicable to plant
P. quinquefolius following harvesting of
P. ginseng to avoid replant diseases in Northeast China. The species
I. robusta has a broad host range, including woody plants and herbs [
11] and contains a higher genetic diversity than
I. mors-panacis, which has a narrow host range, suggesting that the genetics of the pathogen complex could be affected by alternative hosts. Given that various plant hosts are around ginseng fields in Northeast China, host shifts may be frequent. This could increase the possibility that
I. robusta on nearby hosts could mate, which could increase the genetic differentiation of the
I. robusta population.
Sexual reproduction should be another indispensable factor significantly affecting genetic differentiation. The teleomorph has been observed in vitro on
I. robusta [
1,
11] but unknown in
I. communis and
I. mors-panacis [
11]. Although
I. communis had a high frequency than
I. robusta in the present study, more haplotypes were observed in
I. robusta than
I. communis. It suggested that sexual reproduction may accelerate the genetic differentiation of
I. robusta.
This is the first time of using the
his3 gene sequence as a genetic marker in studying the population structure of CLF. Rich sequence data of
his3 in the GenBank allowed us to compare the worldwide populations of
I. robusta and
I. mors-panacis with our isolates. A partial sequence of the
his3 gene with 468 bp generated strong differentiation and a high level of genetic diversity in 169 isolates, suggesting the
his3 gene is informative for population genetic studies in CLF. This is consistent with previous phylogenetic research that the
his3 gene has the highest resolution among the multi loci [
1,
11], which provided the same resolution as four genes combined [
29,
30,
31]. The
his3 marker is not only essential to delineate CLF isolates at species level but also generate finer resolutions of the population within a species. It is common in genetic diversity studies of plants and animals to use
COI gene, but it is rare to use one gene only in fungal genetic diversity research [
32,
33]. We strongly recommend providing
his3 gene sequences in taxonomy and phylogenetic studies of CLF. This method is worthy of application in the genetic study of CLF.