Abstract
Grapevine leaf rust (GLR) in temperate Asia is caused by Neophysopella meliosmae-myrianthae and N. montana; the former is commonly found on commercial grape cultivars (Vitis spp.) and the latter on a wild grape species, Vitis coignetiae. The two GLR fungi were found to co-occur in V. coignetiae at two survey sites in Japan. Under experimental conditions, both fungi parasitize and develop into uredinial and telial stages on commercial grape cultivars and wild species. Despite the assumed involvement of N. montana in GLR symptoms in commercial vineyards, there has been no confirmed report of its incidence, and it is not clear whether N. meliosmae-myrianthae commonly occurs on V. coignetiae under natural conditions. In this study, we aimed to disclose the occurrence and, in particular, the co-occurrence of the two species in a wide array of commercial grape cultivars and V. coignetiae in Japan based on the detection of targeted DNA markers with specific PCR primer pairs. This study confirmed the occurrence of only N. meliosmae-myrianthae infection in symptomatic samples of grape cultivars, while the co-occurrence was observed only in V. coignetiae. Neophysopella montana was widely detected in V. coignetiae specimens.
1. Introduction
Five species are known to be involved in grapevine leaf rust (GLR): Neophysopella meliosmae-myrianthae (Henn. and Shirai) Jing X. Ji and Kakish.; N. montana (Y. Ono and Chatasiri) Jing X. Ji and Kakish.; N. muscadiniae (Buriticá) Jing X. Ji and Kakish.; N. tropicalis Y. Ono, Chatasiri, Pota and Okane; and N. uva (Buriticá and J.F. Hennen) Jing X. Ji and Kakish. (Pucciniales, Basidiomycota). Neophysopella muscadiniae and N. uva are distributed in the Americas, N. tropicalis in Southeast Asia [1,2,3], and Neophysopella meliosmae-myrianthae and N. montana in Japan [4]. Recently, N. meliosmae-myrianthae and N. tropicalis have invaded and firmly established in Brazil [1,5]. Temperate Asian grapevine leaf rust (GLR) is caused by N. meliosmae-myrianthae and N. montana [4,6], with the former commonly found on commercial grape cultivars (Vitis spp.) and the latter on a wild grape species, Vitis coignetiae Pulliat ex Planch. The two morphologically distinct fungi are different in their spermogonial–aecial host preference, i.e., the alternate hosts are Meliosma myriantha Siebold and Zucc. for the former fungus and M. tenuis Maxim. for the latter [4]. Neophysopella meliosmae-myrianthae has been reported to develop uredinial and telial stages on common commercial cultivars such as cv. ‘Delaware’ and cv. ‘Kyoho’ (V. labruscana L.H. Bailey), and wild species such as V. coignetiae, V. thunbergii Siebold and Zucc., and V. flexuosa Thunb. under natural conditions [6]. Neophysopella montana commonly occurs in V. coignetiae in the natural field. The two fungi were found to co-occur in wild V. coignetiae at two survey sites in Tochigi Prefecture, Japan [7]. In the inoculation experiments, both fungi parasitize and develop uredinial and telial stages on commercial grape cultivars, i.e., cv. Delaware and cv. Kyoho, in addition to wild species, i.e., V. amurensis Rupr., V. coignetiae, and V. ficifolia Bunge [4]. Despite the assumed involvement of N. montana in GLR diseases in commercial vineyards, there has been no confirmed report of its incidence. Similarly, it is not clear whether N. meliosmae-myrianthae commonly occurs on V. coignetiae under natural conditions [4,7].
Japan’s table grape production was 48,800 metric tons in 2023, ranking 16th in the world [8]. Japanese viticultural techniques, such as greenhouse forcing, seedless grape cultivation, and the development of various varieties have been uniquely developed, and the technology has been transferred overseas. Grape cultivation is an extremely important part of the Japanese fruit industry [9]. Determining how widespread the two GLR fungi are in both commercial vineyards and natural fields and at what frequencies they occur in diverse grape species is indispensable not only for developing practical and effective GLR management strategies for maintaining high grape quality and stable yields and supply but also for a better understanding of their biological nature and evolutionary relationships. In this study, we aimed to determine the occurrence of populations of the two GLR fungi in a wide array of commercial grape cultivars and the most common V. coignetiae in a whole geographic range of the main island, Honshu, Japan. While surveying a wide range of commercial vineyards in Japan to collect rust specimens for the detection of targeted fungal DNA markers, we also investigated the occurrence of N. montana, which had not previously been confirmed as a parasite of cultivated grapevines in commercial vineyards. We performed this investigation to verify its presence on wild grapevines in the vicinity of the surveyed areas of cultivated grapevine leaf rust.
2. Materials and Methods
Of the rust fungi parasitizing cultivated grape varieties, 34 specimens were collected by the authors, a total of 46 specimens were kept in the Mycological Herbarium of the University of Tsukuba (TSH) (formerly kept in the mycological herbarium of Ibaraki University (IBAR)), a total of two specimens were kept in the Faculty of Agriculture and Life Sciences of Hirosaki University (HHUF), and 21 specimens were provided by collaborators from governmental and prefectural agricultural research stations. In total, 103 specimens originating from 60 locations in 24 prefectures in Japan were examined (Table 1; Supplementary Table S1). The confirmed names of Vitis cultivars are listed in Table 1. The rust fungi parasitizing a wild grapevine, V. coignetiae, included eight specimens collected in this study, 23 specimens from the TSH (formerly kept in the IBAR), 11 preexistent specimens from TSH, and 2 specimens from the HHUF. In total, 44 specimens of rust fungi from wild grapevine leaves originating from fifteen locations in eight prefectures in Japan were examined (Table 2; Supplementary Table S2).
Table 1.
List of the cultivated grapevine samples tested and the result of the DNA detection of the two targeted grapevine leaf rust fungi, Neophysopella meliosmae-myrianthae and N. montana.
Table 2.
List of wild grapevine, Vitis coignetiae, samples tested and the result of the DNA detection of the two targeted grapevine leaf rust fungi, Neophysopella meliosmae-myrianthae and N. montana.
One leaf was arbitrarily selected from the specimens and was sectionalized into six areas, i.e., the left and right parts were further divided into upper, middle, and lower parts, using a metal mesh with square sections measuring approximately 1.2 cm (Figure 1). Spores were scraped from multiple uredinia or telia within a square section of the area using a surgical knife, and DNA extraction was performed. If no uredinium or telium was found in any square sections in the area, and the area was excluded from the analyses. In the case of the cultivated grapevines, DNA was extracted from the spores collected from 531 square sections of 103 specimens (Table 1), and in the case of the wild grapevines, DNA was extracted from spores collected from 166 square sections of 44 specimens (Table 2). DNA extraction was performed according to the methods described by Suyama et al. (1996) [10] and Virtudazo et al. (2001) [11]. In this study, we examined multiple DNA samples from one specimen because some of the specimens collected more than a decade ago were expected to degrade if subjected to this type of analysis.
Figure 1.
An example of selection of uredinia and telia for DNA extraction. A tested leaf was divided into 6 areas, separated by yellow lines (left, right, upper, middle, and lower), and a 1.2 cm square section (red box) where uredinia and/or telia (yellow spots) were present was selected in each area followed by spores sampling.
The PCR of the extracted DNA was performed by using species-specific primers. The primers were designed within the rDNA ITS 2 regions based on the following sequence data, AB354778–AB354789 for N. meliosmae-myrianthae; KC815578–KC815584, KC815598 for N. montana, in this study (Supplementary Figure S1), and then these species’ specificity was rigorously confirmed in advance (Figure 2) to identify the rust fungi. A gradient PCR was performed to determine the optimal annealing temperature. PCR amplification was carried out in 0.2 mL microtubes with the following components: 12.5 µL of Emeral-dAmp® MAX PCR Master Mix (2× Premix), 1.25 µL of N. meliosmae-myrianthae-specific primer Pheuv1 (5′-TGTTGCTGTTACTGGCTCAC-3′ (2 µM)), 1.25 µL of N. montana-specific primer PmontF (5′-CATTGATTACTCTGGTTTATTCCG-3′ (2 µM)), 2.5 µL of primer NL4 [12] (2 µM), 6.5 µL of sterile distilled water, and 1 µL of template DNA. The procedure was performed using a thermal cycler, following the same species-specific primer PCR amplification protocol: initial denaturation at 95 °C for 3 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 60.7 °C for 1 min, extension at 72 °C for 1 min, and a final extension at 72 °C for 10 min.
Figure 2.
The result of the agarose-gel electrophoresis test for species-specific primers designed within the rDNA ITS 2 regions. (A) The test of the Neophysopella meliosmae-myrianthae specific-primer Pheuv1. (B) The test of the N. montana specific-primer PmontF. The primer NL4 (O’Donnell, 1993 [12]) was used as a reverse primer in both of the tests. 1–3: N. meliosmae-myrianthae (880 bp, IBAR 10494, 10485, and 10473, respectively), 4–6: N. montana (740 bp, IBAR 10455, 10463, and 10460, respectively), 7: Pestalotiopsis sp., 8: Aspergillus sp., 9: Colletotrichum sp.1, 10: Colletotrichum sp. 2, isolated from a fruit of grape, 11: Plasmopara viticola, 12: Pestalotiopsis sp. isolated from leaf of grapevine, 13: N. ampelopsidis (on Ampelopsis glandulosa var. heterophylla), 14: N. vitis (on Parthenocissus tricuspidata), 15: SDW, M: DNA marker.
Subsequently, the PCR products were electrophoresed on 1% agarose gel (containing 1× TAE buffer and SYBR Safe DNA Gel Stain, Thermo Fisher Scientific Inc., Waltham, MA, USA) in an electrophoresis tank filled with 1× TAE buffer for 25 min. Nippon Gene OneSTEP Ladder 100 (0.1–2 kbp) (Nippon Gene Co., Ltd., Tokyo, Japan) was used as a DNA size marker. The DNA sequences amplified with this specific method were used to identify the rust fungus/fungi that caused the foliar rust diseases.
3. Results
As a result of the investigation of the rust infection in cultivated grapevines, N. meliosmae-myrianthae markers were detected in 395 DNA samples from 85 specimens collected at 53 locations in 23 prefectures out of 531 DNA samples from 103 specimens collected at 60 locations in 24 prefectures. No N. montana marker was detected in any of the samples (Table 1 and Figure 3). The remaining 135 DNA samples showed no amplification of the target DNA.
Figure 3.
The distribution of DNA markers of Neophysopella meliosmae-myrianthae detected in the rust-infected specimens of Vitis cultivars in 24 prefectures of Japan. Numerals separated by a slash are the numbers of the specimens in which N. meliosmae-myrianthae markers were detected (left) and the total numbers of the rust-infected specimens examined (right). Colored circles indicate the result of DNA marker detection. Neophysopella meliosmae-myrianthae was detected, but N. montana was not.
The samples that bore N. meliosmae-myrianthae sori included 22 specimens of cv. ‘Kyoho’, 8 of cv. ‘Pione’, 5 of cv. ‘Delaware’, and 3 specimens each of cv. ‘Fujiminori’ and cv. ‘Shine Muscat’, as well as one specimen each of cv. ‘Muscat Bailey A’, cv. ‘Queen Nina’, cv. ‘Oriental Star’, cv. ‘Black Beet’, cv. ‘Koshu’, cv. ‘Niagara’, cv. ‘Takatsuma’, cv. ‘Tenshu’, cv. ‘Takao’, cv. ‘Stuben’, cv. ‘Kai Noir’, and cv. ‘Yama Souvenir’.
As a result of the investigation of the rust infection in the wild grapevine V. coignetiae, out of 166 DNA samples from 44 specimens collected at 15 locations in 8 prefectures, infection with either N. meliosmae-myrianthae or N. montana or multiple infections in the same leaf were confirmed by the presence of 46 DNA markers in 20 specimens collected at seven locations in five prefectures (Table 2 and Figure 4). Neophysopella montana-only infection was confirmed by the presence of 31 DNA markers in thirteen specimens collected at a total of five locations in four prefectures, including 4 DNA markers in three specimens from one location in Tochigi Prefecture, 22 DNA markers in seven specimens from two locations in Tottori Prefecture, 4 DNA markers in two specimens from one location in Gunma Prefecture, and 1 DNA marker in one specimen from one location in Aomori Prefecture (Table 2 and Figure 4). Only N. meliosmae-myrianthae infection was confirmed by the presence of 9 DNA markers in three specimens collected at three locations in three prefectures, including 6 DNA markers in one specimen from one location in Niigata Prefecture, two in one specimen from one location in Tochigi Prefecture, and one in one specimen from one location in Nagano Prefecture (Table 2 and Figure 4). Multiple infections by both N. montana and N. meliosmae-myrianthae in the same leaf were confirmed in five specimens from three locations in three prefectures, including one specimen from one location in Tochigi Prefecture (TSH-R58372 = IBAR 10451), three specimens from one location in Tottori Prefecture (TSH-R58385 = IBAR 10464, TSH-R30536, and TSH-R30540), and one specimen from one location in Gunma Prefecture (TSH-R58429 = IBAR 10508) (Table 2 and Figure 4). In addition, 23 specimens showed no amplification of the target DNA. Most of these were collected in the 1900s (Table 2).
Figure 4.
The distribution of DNA markers of Neophysopella species detected in the rust-infected specimens of Vitis coignetiae collected at 15 sites in eight prefectures of Japan. Numerals separated by semicolons are the numbers of specimens in which the DNA markers of Neophysopella species were detected: N. meliosmae-myrianthae markers (left), N. montana markers (center), and markers both of N. meliosmae-myrianthae and N. montana (right). The total number of the rust-infected specimens examined are shown on the left of a slash. Colored circles indicate the result of DNA marker detection.
4. Discussion
In the inoculation experiments conducted by Ono et al. (2012) [4], N. montana was shown to infect two grape cultivars, ‘Kyoho’ and ‘Delaware’. Therefore, it was assumed that grapevine leaf rust on cultivated grapes, which had previously been reported to be caused by N. meliosmae-myrianthae, might also involve N. montana as an additional pathogen. To investigate this interesting phenomenon, we especially focused on GLR in cv. ‘Kyoho’ and cv. ‘Delaware’ in this study. Leaf samples of these cultivars were collected in areas near the distribution range of M. tenuis, the alternate host of N. montana, and tested with PCR analysis to confirm the presence of the rust fungus. As a result, only infections with N. meliosmae-myrianthae were confirmed in all the specimens of commercial grape cultivars tested.
Since Ono et al. (2012) [4] demonstrated that N. montana parasitizes a wild grape species, V. coignetiae, in natural environments, it was speculated that cv. ‘Yama Souvinion’, a grape cultivar derived from a cross between V. coignetiae and cv. ‘Cabernet Sauvignon’, might also be susceptible to N. montana. To investigate this, a survey was conducted in Fukushima Prefecture, especially in areas near the distribution range of M. tenuis. Leaf samples were collected from cv. ‘Yama-Souvinion’ plants showing symptoms of rust, and PCR analysis was performed. The results show that only N. meliosmae-myrianthae DNA was detected. The same approach was applied to cv. ‘Steuben’, cv. ‘Kai Noir’, and an unidentified grape cultivar (rootstock of cv. ‘Kai Noir’) collected at the same site as cv. ‘Yama Souvinion’. As a result, only N. meliosmae-myrianthae was detected.
A comprehensive study undertaken in Australia shows that almost all commercial grape cultivars are moderately to highly susceptible to GLR fungus (identified as N. meliosmae-myrianthae, now N. tropicalis) infection under conditions of an optimum temperature of 25 °C and saturated moisture for 6–12 h [13]. These study results are congruent with an early ecological study in N. meliosmae-myrianthae carried out in Japan [14]. No similar study has been undertaken for N. montana. From the inoculation experiments, however, it is assumed that both GLR fungi possess similar environmental requirements despite their different spermogonial–aecial preference. A possible reason why only N. meliosmae-myrianthae was found in human-managed vineyards might be a matter of geographic distance from a primary inoculum (spermogonial–aecial stages on M. myriantha).
The areas where N. montana was confirmed to occur in wild grapevines overlapped with the distribution of its alternate host, M. tenuis [13]; conversely, the areas where N. meliosmae-myrianthae was confirmed to occur overlapped with the distribution of M. myriantha. According to Horikawa’s distribution map (Supplementary Figure S2), the distribution of M. tenuis overlaps with that of M. myriantha, and both species coexist in some areas. The regions where PCR analysis confirmed multiple infections by both rust fungi (Omineyama, Gunma Prefecture; Yunishikawara, Tochigi Prefecture; Daisen, Tottori Prefecture) are considered to be areas where both rust fungi coexist and may potentially undergo host alternation, as both alternate hosts, M. myriantha and M. tenuis, have been reported (Supplementary Figure S1). In Nikko, Tochigi Prefecture, N. montana infections were found in four specimens of wild grapevines (including co-infections), and N. meliosmae-myrianthae infections were found in one specimen (co-infection). As a result of the testing of specimen TSH-R58372 (=IBAR 10451), PCR amplification products were obtained from two DNA samples, and N. montana DNA was detected in all samples. These results suggest that even in regions where both rust fungi are capable of host alternation, N. montana may have a greater tendency to cause leaf rust and is more likely to parasitize wild grapevines.
We demonstrated that the method using the species-specific primers developed in this study can also be used to detect such infections. While the co-infections of different rust species in wild grapevines were previously reported only in Tochigi Prefecture [7], this study confirmed such infections in Gunma Prefecture (one specimen from one location) and Tottori Prefecture (two specimens from one location). This suggests that co-infection may occur in regions beyond Tochigi Prefecture, as suggested by Ono (2016) [7], and that the coexistence of both rust fungi without clear spatial separation, i.e., without competition, may be possible within a single leaf.
In this study, we were unable to identify the most plausible cause(s) for the failure to detect target DNA in some rusted leaf samples, particularly from V. coignetiae. While this is in part likely due to contamination by other microbes or DNA degradation caused by a long storage period and adverse conditions, most of the specimens of GLR fungi of V. coignetiae, from which the target DNA markers were not amplified, were collected in higher-altitude and/or cold-climate areas, such as Nobeyama (ca. 1350 m ASL; Nagano Prefecture), and at the foot of Mt. Fuji and Lake Yamanakako (ca. 2000 m ASL and 1000 m ASL, respectively; Yamanashi Prefecture) (Table 2 and Figure 4). The possibility, therefore, that the rust populations represented by the samples studied were actually composed of cold-adapted variant subpopulations, having somewhat different DNA sequences that could not be detected with the primer pairs designed in this study, cannot be ruled out. These subpopulations may not necessarily represent a new taxon different from N. montana and N. meliosmae-myrianthae. Further studies would reveal the complex biodiversity of GLR fungi in the wild grape species, V. coignetiae, especially in higher mountainous areas in East Asia.
Contrarily to the results from the experimental inoculations, N. meliosmae-myrianthae was detected exclusively in commercial grape cultivars in all the geographic area surveyed, irrespective of their local climate or altitudinal differences. One of the possible causes of this selective occurrence of N. meliosmae-myrianthae on commercial grape cultivars might be the selective exclusion of N. montana by cultural practices to reduce diseases and pests. Major hosts of N. montana are wild grape species distributed away from commercial vineyards; thus, N. montana populations might be highly susceptible to fungicides, without having been selected for fungicide resistance.
Another possibility is the physical and/or ecological distance (or barriers) between commercial vineyards and the natural habitats of M. tenuis, the alternate host of N. montana. When in the uredinial stage, producing vegetative reproductive urediniospores, GLR fungi do not overwinter on grape canes or on dead grape leaves fallen on the ground [6]. The new infection cycle of N. montana, therefore, must start with the germination of teliospores in dead grape leaves fallen on the ground in the early spring through early summer. Basidiospores produced from the germination of overwintered teliospores subsequently infect and produce aeciospores on M. tenuis. The latter become the inoculum for a new rust fungus infection in grapevines, and the rust spreads from late summer on. Therefore, if M. tenuis is totally absent from the range of V. coignetiae or commercial grape cultivars, the incidence of N. montana rust is unlikely in that geographic range. The competitive exclusion of N. montana by N. meliosmae-myrianthae is not likely, and if it happens, it must be based on a sort of “first come, first served” principle [7]. Supposing that there is a time lag between the two fungi, in their arrival and successful infection in a single susceptible grape leaf, it is probable that the first-comer quickly occupies the susceptible host tissues to abundantly sporulate and that the late-comer cannot establish a new infection because of the little host tissue available. This competition assumes only physical occupation, but not physiological and biochemical relationships. Further experiments with different infection timings are needed to determine whether this sort of competition occurs in GLR. Contrarily, although not well documented in rust fungi, the co-occurrence of two or more rust species, in the same genus or different genera, in the same host individuals and even on the same leaves is a common phenomenon in rust fungi, as exemplified by Ono (2016) [7] and this study in GRL fungi. Ono et al. (2018) [15] explicitly showed that Gymnoconia peckiana (Howe) Trotter var. verrucosa (N. Zhang) J.Y. Zhuang and S.X. Wei, Phragmidium sikangense Petr., and P. barclayi Dietel co-occur in close proximity on a leaf of Rubus erythrocarpus T.T. Yu and L.T. Lu. The co-occurrence or mixed infection of two or more plant pathogens (not restricted to fungal pathogens) is frequently observed, and its synergistic effects on disease incidence and development are widely acknowledged [16,17]. For this reason, any changes in environmental conditions and agricultural management, that might encourage the encounter of the two Neophysopella fungi in intensive vineyards, would facilitate sever GLR incidence.
Supplementary Materials
The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/jof11030193/s1, Figure S1: Sequence alignment of the rDNA ITS2 regions for the design of species-specific primers for Neophysopella meliosmae-myriantha and N. montana. Figure S2: Distribution map of Meliosma myriantha (left) and M. tenuis (right) in Japan (Horikawa, 1972 [18]). Table S1: List of location, geographical coordinate, and collection date of cultivated grapevine samples tested. Table S2: List of locality, geographical coordinate, and collection date of wild grapevine, Vitis coignetiae, samples tested.
Author Contributions
Conceptualization, Y.O. and I.O.; methodology, I.O.; investigation A.K. and I.O.; resources, Y.O., A.K., and I.O.; data curation, A.K. and I.O.; writing—original draft preparation, I.O. and A.K.; writing—review and editing, I.O. and Y.O.; supervision, I.O. All authors have read and agreed to the published version of the manuscript.
Funding
This research received no external funding.
Data Availability Statement
The original contributions of this study are included in the article/Supplementary Materials, and further inquiries can be directed to the corresponding author.
Acknowledgments
We would like to express our gratitude for the following individuals for sampling and supplying specimens: H. Nakamura, Institute of Fruit Tree and Tea Science, National Agriculture and Food Research Organization (present affiliation: Tokyo Office, NIPPON TALC Co., Ltd.); M. Kawaradani, Research Institute of Environment, Agriculture and Fisheries, Osaka Prefecture; N. Kirino, Okayama Prefectural Agriculture, Forestry, and Fisheries Research Center; I. Takahashi, Tenno Branch, Fruit-Tree Experiment Station, Akita Prefectural Agricultural, Forestry, and Fisheries Research Center; S. Nagashima, Shimane Agricultural Technology Center; T. Shinozaki, Ehime Research Institute of Agriculture, Forestry, and Fisheries; M. Otoguro, University of Yamanashi; K. Tanaka, Hirosaki University; K. Sotome, Tottori University, and M. Yamaguchi, Tokyo University of Agriculture. We thank Y. Yamaoka, University of Tsukuba, for his advice on the experiment.
Conflicts of Interest
The authors declare no conflicts of interest.
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