Introduction
Grapevine (Vitis spp.), one of the most economically important and widely grown fruit crops in the world, is used for winemaking, table grapes, and raisin production. However, cultivated grapevines are exposed to various diseases caused by fungi, bacteria, and viruses (Wang et al., 2011). The grapevine leaf rust pathogens were reported as Phakopsora ampelopsidis in Ampelopsis brevipendunculata, Phakopsora vitis in Parthenocissus tricuspidata, and P. euvitis in Vitis spp. (Hiratsuka 1935;Ono 2000). The obligate biotrophic fungus Phakopsora spp., the causal agent of grapevine leaf rust (GLR), is an important disease in grapes in the tropical to temperate areas of the world.
The symptoms of grapevine leaf rust appear on leaves and sometimes on petioles, young shoots, and rachises (Leu, 1988). Small, angular, yellow or brown lesions appear on the adaxial (upper) side and small yellow-orange pustules containing spores, called uredinia, are on the abax-ial (lower) side of the spots in the grapevine leaves infected with pathogens (Nogueira Ju´nior et al., 2017). When the disease is severe, the yellow lesions become necrotic and develop to cover the entire leaf surface. Spores of P. euvitis are known to be transported by wind and air currents (Leu, 1988). The fungus infects young and preferably mature leaves (Naruzawa et al., 2006;Angelotti et al., 2011;Primiano et al., 2017) to affect the normal plant development and reduce the fruit quality and production of grapes in the current and subsequent growing seasons by causing premature leaf fall, consequently reduction of photosynthetic area (Tessmann et al., 2004;Hodson, 2011;Rasera et al., 2019). Although rust disease is controlled by the application of fungicides (Angelotti et al., 2014), the negative impact on the economic costs, the environment, and human health associated with these applications led to search for alternative strategies including the use of the plant’s own defense system against foreign stresses.
As demands for the development of disease-resistant grape cultivars resistant to diseases have increased, wild grapevines have been considered as useful genetic resources resistant to various pathogenic fungi, bacteria, and viruses in grape breeding programs. Ampelopsis brevipedunculata, also known as porcelain berry, is native to northeastern Asia (Rehder, 1940), including Manchuria, Korea, and Japan (Ohwi, 1965). In Asia, A. brevipedunculata has been used in traditional medicine to treat several diseases in the human body and also known to be resistant to diseases and insects. Although there are a lot of studies for the disease of downy mildew and powdery mildew resistance in grapevine, few studies are available for some grapevine diseases such as leaf rust. Despite epidemiological studies on screening of resistance to rust based on disease severity and the number of pustules formed per unit of leaf area in grapevines (Hennessy et al., 2007;Angelotti et al., 2008), there is no report on the expression of genes related to defense response among genotypes against P. euvitis. It is necessary to screening the resistance to rust in Ampelopsis species to provide useful information in selecting genetic resources resistant to rust in grapevine breeding programs. Therefore, in this study, the expression of genes related to defense response was investigated in two genotypes of Ampelopsis species against P. euvitis.
Materials and Method
Plant materials and pathogens
As plant materials, leaves of two genotypes of Ampelopsis brevipedunculata, YG11030 and YG10075, grown in pots at the greenhouse of Yeungnam University, Gyeongsan, Korea, were used for inoculation. Phakopsora euvitis, the causal agent of grape leaf rust, were used for inoculation. Urediniospores of P. euvitis were collected using a mini vacuum cleaner with brushes from naturally infected leaves of grapevines in the vineyard of germplasm in Yeungnam University, Gyeongsan, Korea. Spores were collected from the leaves of various grapevine lines kept as germplasm in the vineyards.
Investigation of Disease Incidence in the Vineyard
The incidence level of the disease was investigated in the vineyard from Sep. to Oct. 2021. The assessment of the incidence level was done by the area covered with the yellow pustules on the abaxial side of the leaves. In this study, the incidence of rust disease was scored in the range from 0 to 5 grades by lesion index. The lesion index represents 0 with no symptom, 1 with 0.1-15% lesion area, 2 with 15-30% lesion area, 3 with 30-50% lesion area, 4 with 50-75% lesion area, and 5 with 75-100% lesion area in the grapevine leaves (Fig. 1).
Evaluation of disease occurrence in vitro and in vivo
The leaves in vitro and in vivo were artificially inoculated with the pathogen and placed for 24 hours and 48 hours in the dark chamber and after that placed under a controlled environment for 10 dai (days after inoculation).
Inoculation of pathogen
Spore suspension (8 × 105spores/ml) of P. euvitis in distilled water was sprayed on the lower surface of the leaves of grapevines for inoculation. Leaves inoculated with the pathogen were then placed in a plastic box containing two layers of moist paper to maintain humidity. Leaves were then incubated in a dark chamber at 25°C and harvested at 24 hours postinoculation (hpi), immediately frozen in liquid nitrogen, and then stored at - 80°C for upcoming analysis.
RNA isolation and real-time polymerase chain reaction (PCR) analysis
Leaf samples were ground in liquid nitrogen using a mortar and pestle. Total RNA was extracted from treated and controlled grapevine leaves using a modified pine tree method (Chang et al., 1993). Total RNA concentration and quality were measured by using a NanoDrop spectrophotometer (NABI, UV spectrophotometer, Korea). Afterward, first-strand cDNA was synthesized from the 500 ng of total RNA using a GoScriptTM Reverse Transcription System (GoScriptTM Reverse Transcription System, Promega, Madison, USA) and next used as a template for PCR. Real-time PCR with template first-strand cDNA from RNA was performed using SYBR Premix Ex (SYBR Premix Ex Taq, TaKaRa Bio Inc., Osaka, Japan). PCR amplification was carried out by subjecting the samples to 95°C for 30 seconds, followed by 40 cycles of 95°C for 5 seconds and 60°C for 30 seconds, and then a melting-curve program at 60°C to 95°C for 5 seconds. The relative transcript levels were calculated using the standard curve method and normalized against the grapevine actin gene (AB372563) as an internal control and melting curves of the amplified products were recorded. All samples were analyzed three times to minimize the experimental error. Nine defense-related genes were tested in real-time PCR and the primer sequences of the genes are shown in Table 1.
Statistical analysis
Significant differences were determined by the Independent (unpaired) sample t-test at P<0.01. All statistical procedures were performed using SPSS 27 for windows (IBM SPSS Inc., Chicago, IL, USA).
Results
Assessments of disease incidence in the field
To assess the rust severity in the leaves of grapevines, the incidence levels of rust were randomly investigated in the vineyard by the lesion index described in Fig. 1. The result of field evaluation showed that there was a significant difference in the rust disease incidence between the two genotypes of Ampelopsis species. There was an incidence level of 2.32 in YG11030 susceptible and 0.53 in YG10075 resistant to rust (Fig. 2). The symptoms of the disease and urediniospores were shown on the lower surface of the leaves (Fig. 3).
In vitro and In vivo evaluation of disease occurrence
In in vitro evaluation, the leaves detached from the plant were artificially inoculated in the laboratory condition and left for 24 hours in the dark chamber. After 24 hours of inoculation, the leaves were placed in light in a controlled environment for 10 days. However, any symptoms did not appear within 10 days.
In in vivo evaluation, the plants that grow in the greenhouse were also artificially inoculated and placed for 48 hours in the dark chamber. After that, the plants are placed in the controlled chamber. After 8 days of inoculation, the yellow spots have appeared on the upper side of the leaves (Fig. 3-A) and then 9-10 (dai) days after inoculation the yellow pustules have appeared on the lower side of the leaves (Fig. 3-B).
Expression of Pathogenesis related genes
To investigate the differential expression of defenserelated genes in grapevine leaves against pathogen infection, the expression of genes was compared in 2 genotypes of grapevines inoculated with P. euvitis. Real-time PCR was performed using primers of nine defense-related genes, which were pathogenesis-related 1 (PR1), β-1,3glutannase (Glu), chitinase (Chi), superoxide dismutases (SOD), glutathione peroxidase (GPX), phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), stilbene synthases 1 (STS1), and resveratrol O-methyltransferase (ROMT).
For the normalization process of real-time PCR, the grapevine actin gene (AB372563) was used. The expression level of tested genes in grapevines inoculated with spores of P. euvitis pathogens is shown in (Fig. 4). As a result of real-time PCR, pathogenesis-related genes including PR1, Glu, and Chi showed up-regulated expression, and the expression level of Glu showed highly significant in YG10075 compared to YG11030 24 hours after inoculation of the pathogen. In both genotypes of Ampelopsis species, the expressions of genes related to reactive oxygen species such as SOD and GPX were up-regulated 24 hours after inoculation of the pathogen. Additionally, the expression levels of SOD and GPX genes were higher in YG10075 than that in YG11030 24 hours after pathogen inoculation. Among the tested genes involved in the phenylpropanoid metabolism, PAL, CHS, and STS genes were up-regulated in both species and their expressions were higher in YG10075 than YG11030. Especially, the expression of the STS1 gene was significantly higher than other genes. However, although ROMT gene expression was upregulated in YG11030, down-regulated in the leaves of YG10075 24 hours after inoculation of the pathogen, which showed a differential expression pattern in defense response against rust pathogen.
Discussion
As grapevine leaf rust is a biotrophic fungus, in this study it has been proved that the pathogen can only survive on the living host. P. euvitis is a biotrophic fungus absorbing nutrients from living cells to have developed infection structures such as haustoria, and limited lytic enzymes. Because they create a nutrient sink to the infection site not to kill host cell, these pathogens induce long-term suppression in host defense (Mendgen and Hahn, 2002).
Plants infected with pathogens exhibit resistant responses by progressing pathogen recognition, signal transduction, and activation of defense responses through various mechanisms to protect themselves. Plants have two major categories of defense mechanisms, R-gene mediated defense and basal defense responses. While basal defense mechanisms provide resistance to a wide range of pathogens, R-gene mediated defense mechanisms provide host resistance and usually respond to specific pathogens (Gururani et al., 2012). Dodds and Rathjen (2010) reported that plants had evolved specialized resistance (R) genes, the products of which can detect the presence of specific effectors and trigger the plant immune system. Grapevine, like other plants, possesses an innate immune system that usually prevents infection by pathogens to express complicated defense mechanisms (Glazebrook, 2005). Active defense mechanisms mostly include the oxidative burst, rapid and localized cell death (hypersensitive response), accumulation of phytoalexins, and synthesis of pathogenesis-related (PR) proteins which possess antimicrobial activities (Berens et al., 2017;Glazebrook, 2005;Jones and Dangl, 2016;Nürnberger and Brunner, 2002;Zhang et al., 2018a).
It was reported that the expression of five PR genes were associated with host-pathogen interactions as the host defense strategies against infection of various Puccinia rust species of wheat (Casassola et al., 2015;Zhang et al., 2018b). There has been reported that PR genes and proteins were induced by pathogen infection in grapevines (Hatmi et al., 2013;Renault et al., 1996). In this study, upregulated expression of the tested PR genes in 24 hours after inoculation revealed that expression of PR genes are involved in defense mechanisms against rust pathogen in tested grapevines.
The generation of ROS in cells is reported to be associated with resistance to stripe rust in the plant-pathogen interaction of wheat, (Wang et al., 2007;Coram et al., 2008). The activity of SOD and GPX was markedly increased after the inoculation of stripe rust in wheat (Chen et al., 2015). There has been described as the accumulation of ROS in grapevine is correlated with a resistant response to pathogen attacks (Carvalho et al., 2015;Oliveira et al., 2009;Singh et al., 2019). In this study, even though the expression of SOD and GPX are not significant, the expressions of both genes in 24 hours after pathogen inoculation were higher than the control, which demonstrates that the expression of SOD and GPX genes might be involved in a role in the defense mechanism in grapevines infected with rust pathogens. It suggests that reactive oxygen species are generated to defend itself against the pathogen in Ampelopsis species infected with GLR.
Phytoalexins has been considered as markers for disease resistance because they possessed biological activity against a wide range of pathogens in plants. In grapevine, the most often observed defense reaction upon fungal infection is the accumulation of phytoalexins, which possess antimicrobial activities against a wide range of pathogens as a subsequent resistant response of plant disease (Pezet et al., 2004, Viret et al., 2018). Stilbenes, major phytoalexins, play an important role in defense grapevine. They are accumulated in leaves in response to infection by pathogens such as P. viticola or B. cinerea. Therefore, consistently with previous reports, in this study, that expression of PAL, CHS and STS genes was induced 24 hours after inoculation of P. euvitis in grapevines responding to the pathogen. However, ROMT has been downregulated 24 hours after inoculation in YG10075. It is considered that ROMT gene with differential expression pattern showed different expression pattern in the synthesis of isoform from resveratrol in 2 genotypes of grapevines responding to the pathogen of grapevine leaf rust.
In this study, there are variations in the expression levels of some tested genes related to defense responses between YG11030 and YG10075, because two genotypes of Ampelopsis species showed the different incidence levels of the disease in on-field assessment. There was significantly high expression of defense-related genes such as PR-genes in moderately resistant genotypes compared to susceptible genotypes of grapevines. Therefore, understanding of plant host response at the molecular level is certainly important for developing novel cultivars in breeding programs for grape resistant to diseases and for preventing the incidence of the diseases in viticulture. The result of this study provides useful information on defense responses and differential expression patterns of defenserelated genes under pathogen infection at the molecular level. The data presented will also be helpful in understanding the mode of the action of defense responses in grapevines to rust disease.
적 요
포도나무 녹병(병원균: Phakopsora euvitis)은 포도생산에서 생산량의 감소와 품질저하를 초래한다. 본 연구에서는 녹병균 을 접종한 포도나무에서 병저항성 반응과 관련된 유전자를 비 교하였다2계통의 개머루 (Ampelopsis sp.)에 포도나무 녹병균 을 접종하고 24시간후에 RNA를 분리하여였으며 pathogenesisrelated 1 (PR1), β-1,3glutannase (Glu), chitinase (Chi), superoxide dismutases (SOD), glutathione peroxidase (GPX), phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), stilbene synthases1 (STS1), 및 resveratrol Omethyltransferase (ROMT) 유전자를 이용하여 real-time PCR 를 수행하였다. YG11030와 YG10075에서 모든 유전자가 발 현되었으며 특히 병원균에 저항성인 YG10075 계통에서 발현 이 많이 유도되었다. Glu와 STS는 YG11030보다 YG10075 에서 발현이 많았다. ROMT 유전자는YG11030에서는 발현이 유도되었으나 YG10075에서는 발현이 감소하였다. 이러한 유 전자의 발현차이는 녹병균 감염에 대한 식물체의 방어반응에 차이가 있음을 의미하는 것으로 이들 유전자는 저항성 반응을 검정하는 데에도 사용이 가능할 것으로 여겨진다. 연구결과는 향후 저항성 반응의 연구와 저항성 품종의 육성에 필요한 정 보를 제공할 것으로 기대된다.
