202 1. Introduction Asparagus (Asparagus officinalis L.), of the family Asparagaceae, is a perennial crop widely cultivated in the world. The crop is cultivated from northern to south- ern regions in Japan, and is an important vegetable. It is expected that production of the crop will increase due to its high profits in many places. The southwestern region of Japan has an advantage because spears emerge earlier in this region than in the northern region. Several dis- eases such as stem blight, leaf spot and brown spot cause serious problems in open field culture in warm regions. Therefore, rain protected culture is necessary (Kobaya- shi and Shinsu, 1990). Among these diseases, stem blight caused by Phomopsis asparagi (Sacc.) is the most seri- ous disease in Japan, Korea, China and Southeast Asia and causes devastating damage to the crop in economi- cally producing fields; the disease is also distributed in Australia, Greece, Italy, New Zealand and USA (Davis, 2001; Elena, 2006; Udayanga et al., 2011). Recently dis- ease damage was also reported in northern parts of Japan (Sonoda, personal communication). The causal fungus first forms small lesions or spots on the lower part of the asparagus stem. The lesions continue to expand and pro- duce yellowish-brown spindle shaped spots (Sonoda et al., 1997). Pycnidia usually appear at the central part of a spot and become secondary sources of infection (Sakai et al., 1992). The symptoms are different with aging of stems, and result in systemic symptom, and finally stems are killed (Fukutomi, 1993). Thus, production of culti- vars with strong resistance to stem blight has become an urgent issue. The genus Asparagus is composed of about 100-300 (Bailey, 1944; Dahlgren et al., 1985) species mainly distributed in arid regions of the Mediterranean, Africa and Asia (Dahlgren et al., 1985). The species are widely used not only as food source but also for medici- nal or ornamental purposes. Sonoda et al. (1997, 2001) reported that there were varietal differences of stem blight resistance but there were no strong resistant culti- vars in A. officinalis. They also reported that several As- paragus species had strong disease resistance, but it was impossible to produce hybrids by interspecific crosses with A. officinalis. Kunitake et al. (1996) produced so- matic hybrids between A. officinalis (2n=2x=20) and A. macowanii (2n=2x=20) by protoplasts electrofusion for the purpose of introduction of disease resistance charac- teristics into asparagus. However, the hybrids failed to mature because of their abnormal genome composition. Marcellán and Camadro (1999) also performed interspe- cific hybridization between A. officinalis and A. densi- florus (Kunth) Jessop cv. Sprengeri (2n=6x=60), but the Stem blight resistance of Asparagus kiusianus and its hybrid with A. officinalis M. Iwato*, M. Kosaza*, Y. Takeuchi*, M. Matsumoto**, M. Inada***, Y. Ozaki**** (1), H. Okubo* * Laboratory of Horticultural Science, Faculty of Agriculture, Kyushu University, Hakozaki, Higashi-Ku, Fukuoka, 812-8581, Japan. ** Institute of Tropical Agriculture, Kyushu University, Hakozaki, Higashi-Ku, Fukuoka, 812-8581, Japan. *** Saga Prefectural Agricultural Experiment Station, Kawasoe, Saga, 840-2205, Japan. **** Laboratory of Agricultural Ecology, Faculty of Agriculture, Kyushu University, Kasuya, Fukuoka, 811-2307, Japan. Key words: Asparagus kiusianus, Asparagus officinalis, disease resistance, interspecific hybrid, Phomopsis asparagi, stem blight. Abstract Resistance of cultivated asparagus (Asparagus officinalis), wild A. kiusianus and their interspecific hybrids to stem blight caused by Phomopsis asparagi (Sacc.) Bubák was evaluated. A. kiusianus had higher resistance than A. of- ficinalis although there were individual variations. The resistance of interspecific hybrids between A. officinalis and A. kiusianus was mostly intermediate, but some individuals had strong resistance equivalent to A. kiusianus. The results suggest that it is possible to introduce the resistance of A. kiusianus to stem blight into A. officinalis through interspecific hybridization for further Asparagus breeding. Adv. Hort. Sci., 2014 28(4): 202-207 (1) Corresponding author: ozaki@farm.kyushu-u.ac.jp Received for publication 17 September 2014 Accepted for publication 6 October 2014 203 endosperm failed to develop normally, leading to subse- quent embryo abortion. Asparagus kiusianus (2n=2x=20) is a dioecious species native to the coastal region of the Japan Sea from Yamagu- chi to northern Kyushu. It is, therefore, expected that the species is adapted with the climate of these regions with high disease or salt resistance. Ito et al. (2011) success- fully produced interspecific hybrids between A. officinalis and A. kiusianus and also obtained backcross progenies. The objective of this study is to demonstrate the use- fulness of A. kiusianus for producing stem blight disease resistant asparagus cultivars, making cultivation in open field culture in southwest region of Japan as well as in other warm climate areas in the world possible. Evaluation of dis- ease resistance was performed in A. kiusianus, cross com- patibility between A. officinalis and A. kiusianus and disease resistance in the interspecific hybrids was surveyed. 2. Materials and Methods Plant materials Asparagus officinalis cv. UC157F 1 , as the control cul- tivar (Sonoda et al., 1997), and A. kiusianus Keya line from Fukuoka, Japan were used for inoculation tests in 2010 to compare the resistance to stem blight in these two species (Table 1). UC157F 1 and A. kiusianus (Nijino- matsubara line from Saga, Japan) were used for crossing experiments. UC157F 1 and its intraspecific F 1 hybrids, A. kiusianus (Nijinomatsubara line from Saga, Keya line and Uminonakamichi line from Fukuoka, Japan) and interspe- cific F 1 hybrids of A. officinalis with A. kiusianus (OK) were used for inoculation tests in 2011 and 2012 (Table 1). Inoculum Phomopsis asparagi strain P1, preserved in Saga Pre- fectural Agricultural Experiment Station, was cultured on potato sucrose agar (PSA) medium at 25°C for one month. After sporulation, conidiospores were suspended in steri- lized water and adjusted to the inoculum density of 2×106 spores/ml for use in the following tests. Interspecific cross Interspecific crosses between A. officinalis (female) and A. kiusianus (male) and intraspecific cross in A. of- ficinalis cv. UC157F 1 were made by hand pollination in a greenhouse of Kyushu University Farm in 2010. Obtained seeds were germinated at 25°C in plastic petri dishes, and germinated seeds were sown in vermiculite in plastic cell trays in an incubator. They were transplanted in plastic pots six months after seed sowing and transferred. Geno- mic DNA was extracted from young cladodes of each plant by modified CTAB method (Stajner et al., 2002). Hybrid- ity of interspecific progenies was confirmed by three SSR markers (Caruso et al., 2008), AG2, AG7 and AG10, fol- lowed by Takeuchi et al. (2012). Evaluation of stem blight resistance for inoculation test Inoculation tests were performed three times from November 2010 to January 2011 with UC157F 1 and A. kiusianus, Keya line grown in plastic pots. Seven to 10 plants were used for each test. The plants were inocu- lated with the vinyl tape and cotton method by reference to the protocols previously described by Sonoda et al. (1997, 1999, 2001). Absorbent cotton moistened with spore suspension was put around the basal part (2-5 cm below the lowest branching nodes) of the stem, and cov- ered with vinyl tape. The plants were maintained at 25°C with 90% humidity for three days, followed by incuba- tion at 25°C and at 60-70% humidity in a growth cham- ber. The spears, which emerged two to three weeks after cutting the areal stems of the seedlings, were examined. Disease severity was determined by scoring 0-4 grades for each plant weekly until five weeks after inoculation. The disease severity grade (DSG) was: 0 = no lesion; 1 = small-sized lesion (<1 cm in length); 2 = spread lesion; 3 = large-sized lesion (>half of the aerial stem) or defolia- tion; 4 = aerial part death. Percentage of infected plants and disease indices were calculated, as follows: Percentage of infected plants = Number of infected plants Total number of plants employed × 100 Disease index (DI) = ∑(Number of plants classified into each grade × DSG number) Total number of plants employed × 4 × 100 The degree of pycnidia formation was also surveyed. Resistance in UC157F 1 and its intraspecific F 1 hybrids, A. kiusianus (Keya line, Nijinomatsubara line and Umi- nonakamichi line) and interspecific F 1 hybrids between them was evaluated by inoculation test three times from August to September and from November to December in 2011 and from October to November in 2012. Assess- ments were performed according to the procedure as de- scribed above. Table 1 - Plant materials used for inoculation tests Species Cultivar and line Tested year No. of individuals investigated A. officinalis UC157F 1 2010 14 A. officinalis UC157F 1 , F 1 progenies of ‘ UC157F 1 ’ 2011, 2012 25 A. kiusianus Keya line 2010 15 A. kiusianus Keya line, Uminonakamichi line, Nijinomatsubara line 2011, 2012 21 Interspecific hybrid Hybrids between A. officinalis and A. kiusianus 2011, 2012 25 204 3. Results Evaluation of the resistance to stem blight in A. officinalis and A. kiusianus Most of the plants in both species showed pathogenic symptoms on the stems (Table 2). All the inoculated as- paragus cultivars began to show blight by one week af- ter inoculation and the aerial parts of all plants were dead within five weeks. In contrast, A. kiusianus showed lower DI than A. officinalis and more than half of the plants sur- vived five weeks after inoculation although they exhibited symptoms. There were differences of susceptibility among A. kiusianus accessions, whereas all A. officinalis acces- sions showed high susceptibility with severe symptoms. Pycnidia formation was frequently observed in A. officina- lis from two to four weeks after inoculation (data are not shown). A few accessions showed pycnidia formation in A. kiusianus, whereas some other individuals showed no symptoms possessing strong resistance. Interspecific crosses between A. officinalis and A. kiusianus Fruit set rate and number of seeds in interspecific crosses between A. officinalis (female) and A. kiusianus (male) were lower than those in intraspecific crosses among A. officina- lis (Table 3). Germination rate of the interspecific hybrid was high (76.1%) at 25°C. Hybridity of the progenies was confirmed by three SSR markers (Caruso et al. 2008), AG2, AG7 and AG10 (data are not shown). The hybrid plants showed intermediate morphology between the parents. Evaluation of the resistance to stem blight in the interspe- cific hybrids Most of the hybrid plants and their parents showed pathogenic symptoms (Fig. 1, Table 4). The aerial stems of all inoculated A. officinalis showed blight by one week af- ter inoculation and most of them faded within four weeks (Fig. 2 D). In contrast, the aerial stems of A. kiusianus and the interspecific hybrids survived even four weeks after Table 2 - Development of disease symptoms in A. officinalis and A. kiusianus inoculated with P. asparagi Species Percentage of infected plants Disease index 1 (z) 2 3 4 5 1 2 3 4 5 A. officinalis 100 a 100 a 100 a 100 a 100 a 45 a 54 a 88 a 98 a 100 a A. kiusianus 77 a 85 a 93 a 93 a 93 a 25 b 38 b 54 b 63 b 69 b (z) Weeks after inoculation. Different letters in a column indicate significant difference according to student’s t-test (P≦0.05). Table 3 - Result of interspecific crosses between A. officinalis and A. kiusianus Seed parent Pollen parent No. of pollinated flowers No. of obtained fruits (%)(z) No. of obtained seeds (n) (y) No. of germinated seeds (%)(x) No. of obtained seedlings A. officinalis × A. kiusianus 301 27 (9.0) 67 (2.5) 51 (76.1) 42 A. officinalis × A. officinalis 46 25 (54.3) 119 (4.8) 104 (87.4) 45 (z) No. of obtained fruits/No. of pollinated flowers)×100. (y) No. of obtained seeds/No. of obtained fruits. (x) (No. of germinated seeds/No. of obtained seeds)×100. Fig. 1 - Frequency of individuals with their disease severity grades (DSG) in A. officinalis, A. kiusianus and F 1 hy- brids screened for resistance to stem blight. Tests were performed in August (A) and November (B) in 2011.  0 = no lesion; n 1 = small-sized lesion (<1 cm); n 2 = spread lesion; n 3 = large-sized lesion (>half the size of plant) or defoliation; n 4 = aerial part death. 205 the inoculation (Fig. 2 E, F). The DI’s in A. kiusianus and the interspecific hybrids were lower than those in A. of- ficinalis although lesion was formed in most of the plants (Table 4). The disease severity grade of A. officinalis was high compared with that of A. kiusianus and the interspe- cific hybrids (Fig. 1). Most A. kiusianus showed lower grades even five weeks later. The interspecific hybrids showed varied degrees of symptoms and the distribution of DSG was intermediate between the parental species. Diseased lesions were observed in all the stems of A. of- ficinalis and numbers of pycnidia were frequently formed four weeks after inoculation (Figs. 3, 4). There was little pycnidia formation in A. kiusianus and interspecific hy- brids, while lesions were observed in the stems of most plants of A. kiusianus and the interspecific hybrids. The hybrids showed intermediate susceptibility between their parent species and their pycnidia formation rates were as low as the degree of A. kiusianus (Fig. 4). Table 4 - Development of disease symptoms in A. officinalis, A. kiusianus and their hybrids inoculated with P. asparagi Species Percentage of infected plants Disease index 1 (z) 2 3 4 5 1 2 3 4 5 A. officinalis 95 a 100 a 100 a 100 a 100 a 30 a 35 a 60 a 90 a 100 a A. kiusianus 81 a 88 a 88 a 88 a 88 a 27 a 31 a 34 b 37 b 40 b Hybrids 86 a 92 a 92 a 92 a 95 a 29 a 36 a 40 ab 47 b 59 b (z) Weeks after inoculation. Different letters in a column indicate significant difference according to Turkey’s multiple range test (P≦0.05). Fig. 2 - Disease symptoms of A. officinalis (A, D), A. kiusianus (B, E) and F 1 hybrids (C, F) inoculated with P. asparagi. A-C: 1 week after inoculation, D-F: 4 weeks after inoculation. Scale bars 9 cm. Fig. 3 - Lesion on stems of A. officinalis (A), A. kiusianus (B) and in- terspecific hybrids (C, D) inoculated with P. asparagi. Arrows indicate pycnidia. Scale bars 1 mm. Fig. 4 - Rates of lesion and pycnidia formation of A. officinalis, A. kiu- sianus and interspecific hybrids four weeks after inoculation with P. asparagi. Bars represent SE (n=3). Different letters indicate significant difference according to Turkey’s multiple range test (P≦0.05). 206 4. Discussion and Conclusions The symptoms were severer in this study than those re- ported by Sonoda et al. (1997, 1999, 2001). DI was nearly 100 in A. officinalis four weeks after inoculation in this study, while it ranged from 42 to 79 in another investiga- tion (Sonoda et al., 1997). This might be due to aggressive pathogenicity of the phomopsis strain used or differences in environmental conditions from the previous study (So- noda et al., 1997). The results of the present study show that A. kiusianus had higher resistance than A. officina- lis with various degrees of resistance among individuals. Although the lesions were observed in most A. kiusianus plants, the resistant accessions displayed reduced disease development and varying levels of resistance. Pycnidia were infrequently formed on the stems of A. kiusianus, restricting the spread the pathogen. It is thought of as in- complete (or partial) resistance (Poland et al., 2009). The rate-reducing resistance or partial resistance is believed to be effective against a large number of pathogen genotypes and durable resistance since it is non-race specific (Niks and Rubiales, 2002; Poland et al., 2009). Therefore, the resistance identified in A. kiusianus will be useful for as- paragus breeding programs. There were differences in disease resistance among As- paragus species (Sonoda et al., 2001). Asparagus densiflo- rus, A. virugatus, A. asparagoides and A. macowanii were highly resistant. They are, however, genetically distant to A. officinalis and extremely difficult to obtain viable inter- specific hybrids. 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