Impaginato 543 Adv. Hort. Sci., 2019 33(4): 543­552 DOI: 10.13128/ahsc­8187 Study on relationship between morphological and physiological traits with resistance to rust fungus (Puccinia allii) in Iranian garlic clones A. Anjomshoaa 1, H. Jafary 2, M.R. Hassandokht 3 (*), M. Taheri 2 , V. Abdossi 1 1 Department of Horticulture Science and Agronomy, Science and Research Branch Tehran, Islamic Azad University, Iran. 2 Plant Protection Research Department, Zanjan Agricultural and Natural Resources Research and Education Center, AREEO, Zanjan, Iran. 3 University College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran. Key words: Allium sativum L., garlic rust, combined variance analysis, infection frequency. Abstract: In the present study we collected 12 clones of garlic from different geographical origin in Iran. The clones were sown in a field trial under natural infection of the rust fungus during two consecutive years. After 210 days, the reactions of the clones to the disease as well as the morphological features of the clones were evaluated. The results of analysis of variance on morphological traits showed a significant difference among the clones in terms of bulb weight, mean clove weight, number of bulb skin, number of cloves in the bulb, leaf temperature and the percentage of clove dry weight, and nutrient uptake for N,P,K, Mn and Zn. The results showed a positive and significant correlation between the leaf temperature, photosynthesis, nitrogen and manganese uptake and percentage of leaf infection at 1% probability level. The results of the infection frequency showed that the clones ‘Gilvan1’ and ‘Lalejin’ had the lowest percentage of infection and were identified as resistant clones to the rust disease. The results also showed that garlic clones reacted differently to the rust fungus and are separated into resistant, semi resistant, semi­suscepti­ ble and susceptible clones. 1. Introduction Garlic (Allium sativum L.) is one of the most important vegetable crops in the world. Asian countries such as China, India, Afghanistan and Iran are known as the main growing area of this vegetable. Currently, garlic cultivation and production has been developed with a wide variety from East Asia to South America. Due to the commercial, economic and phar­ maceutical­industrial importance of garlic, the interest in increasing pro­ (*) Corresponding author: mrhassan@ut.ac.ir Citation: ANJOMSHOAA A., JAFARY H., REZA HASSAN­ DOKHT M., TAHERI M., ABDOSSI V., 2019 ­ Study on relationship between morphological and phy‐ siological traits with resistance to rust fungus (Puccinia allii) in Iranian garlic clones. ­ Adv. Hort. Sci., 33(4): 543­552. Copyright: © 2019 Anjomshoaa A., Jafary H., Reza Hassandokht M., Taheri M., Abdossi V. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 29 August 2019 Accepted for publication 9 September 2019 AHS Advances in Horticultural Science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2019 33(4): 543­552 544 duction, yield and development of new varieties is rapidly growing (Cunha et al., 2012). The world aver­ age yield of garlic in the last 20 years has been around 10 tons per hectare, while in recent years it increased to 18 tons per hectare (FAOSTATS, 2018). The Iranian plateau is one of the most important cen­ ters of vegetation diversity in the world (Mousavi, 1994) and among scientists, it is known as the arc of garlic distribution. Morphological and molecular studies have indicated a wide variety of Iranian garlic clones distributed all over the country (Baghalian et al., 2005; Vafaee, 2007). Despite the long history of forced apomixis in the garlic, its cultivars show a great diversity of morphological, physiological and biochemical differences (Etoh and Simon, 2002; Ammarellou et al., 2014). Garlic colonies have a wide variety in vegetative traits, taste and flavor, bolting and fertility capacity. The cultivar characteristics dif­ fer significantly according to the location of cultiva­ tion and climate, and the weather conditions has a significant effect on cloves, flowering and taste of garlic. All garlic colonies are sterile, and therefore genetic variation in this plant may only be due to ran­ dom mutations or non­sexual variation and to the introduction of new genetic variation using modern molecular techniques. Biochemical and molecular studies indicate that the highest level of heterogene­ sity occurs within the genetic reserves of central Asia, which may include favorite genes for use in future genetic studies, as well as the improvement of plant programs. Kallo and Bergh stated that cloves’ mor­ phological traits, in particular number and size of leaves, height of the flowering stem, number of bulb shells, reaction to temperature, seed dormancy, quality of maintenance, vegetative and maturity peri­ od duration, are genetically controlled and these are polygenic traits (Kallo and Bergh, 1993). One of the most important problems faced by farmers in relation to garlic cultivation is the out­ break of fungal rust disease due to high humidity of cultivated areas. Most herbaceous fungi are destroyed by fungicides, but for many of them there is still no proper and effective fungicide. In addition, the use of fungicides generally causes pollution of the environment, groundwater resources, and prod­ ucts themselves. The cause of garlic rust is the fungus Puccinia allii that is an airborne fungus, and its release of spores is very fast (Michael and Sarah, 1995); spores are commonly transported by the wind and causes the disease characterized by a sore or pustules at the leaf level. The teliospores live in non­ crop seasons in the soil and plant infected residuals. The disease develops at temperatures below 10 °C and is destroyed by frozen dew. If the garlic is sub­ jected to drought stress or excess water or con­ densed due to excessive consumption of nitrogen fertilizer, it will become susceptible to the disease (Houshiarfard and Pourabdollah, 2015). In the world, an overgrowth of rust on garlic has been reported in many cases (Schwartz and Mohan, 1999). For example, garlic rust is one of the major problems in garlic cultivation in the United States. Studies show that the clones do not have complete resistance to garlic rust, they may be tolerant showing (on average) 51% reduction in the product (Coviello, 2007). Selection of resistant clones from the genetic complex of Iranian local clones is one of the pre­ breeding research priorities of the country in Iran. Identification of resistant crop cultivars has many advantages over the use of chemicals and other con­ trol methods. Since the economic importance of garlic rust has been grown up in recent years, few studies have been focused on the identification of morpho­ logical traits of garlic related to the yield, but none of the studies addressed on the relationship between morphological, physiological component and the resistance to the rust fungus. This study aimed to investigate the physiological and morphological fea­ tures in the resistant and the susceptible Iranian garlic clones, with the ultimate scope to achieve resistant colonies to the garlic rust in selected clones. 2. Materials and Methods Plant materials Based on geographical distribution of garlic in Iran, we indicated the main cultivation regions with long crop history throughout the country. In each region, we collected one sample which represented the main geographical characteristic of that region to avoid any doubling in sampling and to collect as much as distinct clones. In total we collected 12 superior clones from all over the country (Table 1). The cloves were planted after disinfection with fungi­ cide carbendazim and confidor insecticide in a field trial in Chavarzagh Tarom Zanjan (48 46’ 43.34” E ­ 36 59’ 51.33” N) and altitude of 484 meter from sea level and temperate­semi­arid climate in a complete­ ly randomized block design with three replications during two consecutive years. Soil texture of cultiva­ tion site was clay loam with pH= 7.6 and electrical conductivity of 0.8 dS/m, percentage of neutralizing matter was 6.2%, and available phosphorus and Anjomshoaa et al. ‐ Iranian garlic resistance to rust fungus 545 potassium in the soil were 3.8 and 261 mg/kg, respectively. In the annual cultivation process, the cloves of each clone were grown on 35 cm stacks with cultivation depth of 3 cm and 10 cm spacing. Due to possible genetic variation on resistance to the rust fungus within each clone, the most resistant seedlings of clones were selected for diseases resis­ tance evaluation in the second year. At garlic harvest­ ing stage, the bulbs were harvested at the time of physiological maturity, when the leaves fell and 70% dried (Rubatzky and Yamaguchi, 1997). Evaluation of morphological traits The morphological traits were evaluated accord­ ing to the descriptor recommended by the International Plant Genetic Resources Institute (IPGRI, 2000). The evaluated morphological traits included bulb weight, bulb diameter, number of leaves, mean clove weight, number of bulb sheet, number of cloves in a bulb, color of clove, bulb and leaf, side view of bulb, shape of mature bulb and type of bulb structure. Leaf color and number were evaluated when the plants were fully developed, while the traits of bulb were studied after complete maturation of the plant and bulb harvesting. Evaluation of physiological traits At the end of March each year, the leaf chloro­ phyll content was estimated by measuring 10 leaf samples per replication and estimation of the mean data was done with the help of SPAD device. Other physiological measurements on leaf such as pure photosynthesis, stomatal conductance of H2O, leaf surface temperature and transpiration sub stomatal CO2 at the full leaf growth stage were performed by UK manufacturing ADC device (ADC BioScientific LCi Analyser Serial No. 32648). Sampling of green leaves was done at full leaf growth stage to estimate the rate of nutrients absorption and dry matter percent­ age of the leaf and immediately transferred to labo­ ratory of Zanjan Agricultural Research Center. The percentage of dry weight of cloves and leaves was calculated and recorded by placing a sample of cloves or leaves of each treatment for 24 hours in oven at 70°C and measuring the weight of samples before and after drying. The nutrient uptake was evaluated in Water and Soil laboratory of Zanjan Agriculture Research Center. After sampling the plant leaves, they were immediately transferred to the laboratory and after washing they were dried immediately in a 70° C oven for 48 hours. Then, the samples were digested with acid and the plant extract was pre­ pared. Finally, macro elements such as nitrogen, potassium, phosphorus and micro elements such as zinc, copper and manganese were measured by atomic absorption and calorimetric (Emami, 1996). Evaluation of resistance to the rust fungus In order to evaluate the frequency of leaf infection by rust fungus among the clones, the numbers of pus­ tules were recorded at 4 stages (20 April, 5 May, 20 May and 5 June). The evaluation method of infection frequency of the disease was as follows: 10 plants per plot were randomly selected. The average of number of pustules on leaves of 10 plants was determined as the percentage of rust infection in each clone (Clifford and Jones, 1983; Dhingra and Sinclair, 1995). Methods and tools for analyzing the data In this study, statistical software SAS and SPSS was used to analyze the statistical data. Analysis of vari­ ance of traits and their mean comparison were done using Duncan test at 1% level. The correlation between traits was measured by Pearson’s two­ Table 1 ­ Origin specifications of Iranian garlic clones Row Clone Country Province City/Region Longitude Latitude Altitude 1 Dezfol Iran Khozestan Dezfol 48° 25' 52.57" E 32° 22' 59. 08" N 143 3 Sahneh Iran Kermanshah Sahneh 47° 41' 41.08" E 34° 28' 26. 53" N 1354 4 Lalejin Iran Hamedan Lalejin 48° 28' 34.42" E 34° 58' 25. 22" N 1700 5 Azar shahr Iran Azarbayejan sharghy Azar shahr 45° 58' 59.78" E 37° 44' 1. 64" N 1415 6 Lahijan Iran Gilan Lahijan 50° 0' 12.07" E 37° 12' 25. 46" N 4 7 Khaf Iran Khorasan razavi Khaf 60° 8' 50.48" E 34° 34' 18. 53" N 975 8 Sojas Iran Zanjan Sojas 48° 33' 04.45" E 36° 14' 24. 14" N 1774 9 Hesar Iran Zanjan Hesar 47° 43' 7.24" E 36° 57' 26. 62" N 1158 10 Gilvan 1 Iran Zanjan Gilvan 1 49° 4' 48.88" E 36° 48' 10. 53" N 340 11 Gilvan 2 Iran Zanjan Gilvan 2 49° 7' 51.54" E 36° 47' 10. 94" N 340 12 Chavarzagh Iran Zanjan Chavarzagh 48° 46' 43.34" E 36° 59' 51. 31" N 484 Adv. Hort. Sci., 2019 33(4): 543­552 546 domain test with the SPSS software. At the end of the second year, combined variance analysis was per­ formed. 3. Results Analysis of variance The results of two­year combined analysis of vari­ ance of morphological traits showed a significant dif­ ference between the clones in terms of bulb weight, mean clove weight, number of bulb skin, number of cloves in the bulb at 1% level, while there was no sig­ nificant difference between the bulb diameter and number of leaves (Table 2). The results of combined analysis of variance for two years from physiological traits showed a signifi­ cant difference between the studied clones in leaf surface temperature and clove dry weight percent­ age at 1% probability level. In terms of stomatal con­ ductance and photosynthesis, the difference was sig­ nificant at 5% probability level, while there was no significant difference between substomatal CO2, tran­ spiration, chlorophyll and leaf dry weight percentage among the clones (Table 3). Combined analysis of variance in terms of nutrient uptake of N, P, K, Mn, and Zn showed that a signifi­ cant difference was found between the clones at 1% level and in terms of Cu uptake at 5% probability level (Table 4). The results of combined analysis of variance in terms of the resistance of the clones to the rust fun­ Table 2 ­ Results of combined analysis of variance for morphological characteristics in some Iranian garlic clones S.O.V df MS Bulb weight Bulb diameter Mean clove weight Number of bulb skin Number of cloves bulb Leaf number Year 1 11295.04 ** 5.24 * 2.41 NS 0.12 NS 0.05 NS 0.00 NS Rep/Year 4 120.94 0.46 1.68 0.06 3.81 0.44 Treat 11 351.53 ** 0.59 NS 5.77 ** 22.31** 32.77 ** 0.46 NS Treat × Year 11 363.21 ** 0.57 NS 0.53 NS 0.42 NS 0.38 NS 0.00 NS Error 44 103.00 0.57 0.95 0.87 1.84 0.56 CV% 17.48 15.30 25.46 18.73 11.41 10.83 Table 3 ­ Results of combined analysis of variance for physiological characteristics in some Iranian garlic clones *, **, NS= significant at 5%, 1% and not significant probability levels, respectively. *, **, NS= significant at 5%, 1% and not significant probability levels, respectively. S.O.V df MS Clove dry weight Leaf dry weight Chlorophyl Sub stomatal CO2 Stomatal conductance of H2O Photosynthesis Transpiration Leaf surface temperature Year 1 590.64 ** 0.61 NS 123.76 NS 9531.20 NS 0.0013 NS 233.24 ** 160.68 ** 894.64 ** Rep/Year 4 0.21 1.84 39.75 2517.86 0.0036 0.37 1.70 7.26 Treat 11 8.55 ** 1.89 NS 39.51 NS 455.23 NS 0.0032 * 10.53 * 0.38 NS 3.14 ** Treat × Year 11 13.66 ** 1.44 NS 62.30 NS 1061.43 * 0.0032 * 3.74 NS 0.58 NS 1.74 ** Error 44 0.84 2.48 39.16 581.20 0.001 5.21 0.51 0.52 CV% 3.11 12.29 11.18 10.05 17.50 19.38 11.23 1.96 Table 4 ­ Results of combined analysis of variance for physiological characteristics in some Iranian garlic clones S.O.V df MS Nitrogen Phosphorus Potassium Manganese Zinc Copper Year 1 26/21 ** 0/026 NS 0/0004 NS 445/80 NS 3786/84 ** 1/85 NS Rep/Year 4 0/064 0/009 0/199 25/19 32/23 2.45 Treat 11 0/24 ** 0/0071 ** 1/326 ** 67/36 ** 56/87 ** 1/28 * Treat × Year 11 0/098 ** 0/0075 NS 0/429 ** 22/86 NS 15/60 NS 2/53 ** Error 44 0/017 0/0006 0/09 15/32 9.81 0/61 CV% 2.9 7.31 7.32 10.27 11.08 9.39 *, **, NS= significant at 5%, 1% and not significant probability levels, respectively. Anjomshoaa et al. ‐ Iranian garlic resistance to rust fungus 547 among the physiological traits was related to photo­ synthesis (19.38). Among the morphological traits, the highest coefficient of variation was related to the mean weight of the clove (25.46). In the study of the percentage of infection of garlic to the rust fungus, the highest coefficient of variation appeared at the second stage of evaluation in the early May (33.60). Regarding to significance of analysis of variance among the clones, for some traits the mean compari­ son was done using Duncan’s multi­domain test. The results of the two­year mean comparison of morpho­ logical traits showed that among the studied clones, the lowest weight was related to the clones ‘Chavarzagh’ (49.84) and ‘Lahijan’ (51.47) and the high­ est was related to the clones ‘Sahne’ (79.40) and ‘Sojas’ (61.11). In terms of the mean weight of the clove, the lowest weight belonged to the clone ‘Khaf’(1.97) and the highest weight belonged to the clones ‘Sahne’ (5.14) and ‘Gilvan1’ (4.98). The mean comparison of the studied clones showed that the lowest number of bulb skin was for the clones ‘Chavarzagh’ and ‘Lahijan’ (2.33) and the highest was for the clone ‘Azar Shahr’ (7.16). In terms of number of cloves in a bulb, the lowest number was related to the clone ‘Hesar’ (8.66) and the highest was related to the clone ‘Khaf (17.33) (Table 6). gus at four stages of measurement showed that the difference at the first stage was significant at 1% level. At the next three stages, the difference in resis­ tance between the clones was significant at 5% level (Table 5). The effect of year was also significant in terms of the leaf surface temperature, photosynthesis, tran­ spiration, percentage of clove dry weight, absorption of nutrients Zn and N and bulb weights at 1% level. In term of resistance to garlic rust, the effect of the year was significant at 4 stages at 1% level. The effect of year on the treatment showed that the leaf surface temperature, N, P, K and copper nutrient uptake percentage, percentage of dry weight cloves, bulb weight, and percentage of resistance gar­ lic rust were significant at the first and second stages of growth at 1% level and substomatal CO2, stomatal conductance, and percentage of infection of garlic rust were significant at the 3rd and 4th stages of growth. Other physiological traits including photosyn­ thesis, transpiration, chlorophyll content, dry matter percentage, manganese and zinc intake, and morpho­ logical traits including the bulb diameter, mean bulb weight, number of the bulb skin, number of cloves in the bulb and number of leaves did not showed signifi­ cant differences. The highest coefficient of variation Table 5 ­ Results of combined analysis of variance for resistance to rust in some Iranian garlic clones S.O.V df MS 20/04/2019 05/05/2019 20/05/2019 05/06/2019 Year 1 491.42 ** 651.86 ** 2604.74 ** 2947.88 ** Rep. Year 4 0.35 4.65 24.94 11.23 Treat 11 1.38 ** 15.19 * 75.02 * 144.83 * Treat × Year 11 1.38 ** 20.63 ** 84.50 * 119.35 * Error 43 0.35 6.29 44.57 63.58 CV% 22.85 33.60 32.58 20.29 Table 6 ­ Mean comparison of morphological traits in some Iranian garlic at two years *, **, NS= significant at 5%, 1% and not significant probability levels, respectively. Treatment Bulb weight (gr) Bulb diameter (mm) Mean clove weight (gr) Number of bulb skin Number of cloves per bulb Leaf number per plant Gilvan 1 60.13 b 4.96 a 4.98 a 5.83 abc 12.00 cd 6.66 a Gilvan 2 56.65 b 5.07 a 4.69 a 4.83 c 12.33 bc 6.66 a Azar shahr 55.08 b 5.05 a 3.15 bcd 7.16 ab 13.16 bc 6.66 a Hesar 52.77 b 4.77 a 3.85 abc 5.66 bc 8.66 f 7.33 a Lahijan 51.47 b 4.96 a 3.68 abc 2.33 d 12.00 cd 7.33 a Khor va biabanak 54.22 b 4.88 a 4.07 ab 2.83 d 11.33 cde 7.33 a Sojas 61.11 b 5.27 a 2.28 cd 6.66 ab 9.66 def 6.66 a Dezfol 56.41 b 4.80 a 4.47 ab 2.66 d 14.33 b 7.00 a Sahneh 79.40 a 5.36 a 5.14 a 6.33 abc 11.50 cde 7.00 a Khaf 58.45 b 4.10 a 1.97 d 5.83 abc 17.33 a 7.00 a Chavarzagh 49.84 b 4.93 a 3.89 abc 2.33 d 11.33 cde 7.00 a Lalejin 60.85 b 5.08 a 3.94 abc 7.33 a 9.33 ef 6.66 a In each column different letters mean significant differences between samples. 548 Adv. Hort. Sci., 2019 33(4): 543­552 The results of the two­year mean comparison of physiological traits showed that among the studied clones in terms of the leaf surface temperature, the clone ‘Gilvan1’ had the highest leaf surface tempera­ ture (37.98 ℃) while the clone ‘Azar shahr’ had the lowest one (35.53 ℃). Stomatal conductance was highest in the clone ‘Azar shahr’ (0.23) while the low­ est value was found for the clone ‘Gilvan1’ (0.15). The clone ‘Azar shahr’ had the highest photosynthe­ sis rate (13.35) while the clone ‘Gilvan1’ had the low­ est one (8.80). In terms of percentage of the clove dry weight, the highest was related to the clone ‘Lahijan’ (31.92) and the lowest value was related to the clone ‘Azar shahr’ (28.11) (Table 7). The mean comparison of the studied clones in terms of nutrient uptake showed that the highest nitrogen uptake was related to the clones ‘Dezful’ (4.83) and ‘Chavarzagh’ (4.77) and the lowest was related to the clones ‘Hesar’ (4.22) and ‘Khaf’ (14.32). In terms of phosphorus uptake, the highest uptake was related to the clone ‘Lalejin’ (0.44) and the low­ est was related to the clone ‘Sojas’ (0.30). In terms of potassium and manganese uptake, the highest uptake was related to the clone ‘Dezful’ in potassium (4.85) and manganese (42.77), and the lowest was related to the clone ‘Lalejin’ in potassium (3.62) and manganese (30.89). In terms of zinc and copper uptake, the highest uptake was related to the clone ‘Lalejin’ in zinc (35.78) and copper (9.21), the lowest zinc uptake was for the clone ‘Gilvan1’ (24.61) and the lowest copper uptake was for the clone ‘Khaf’ (7.55) (Table 8). It is worth noting that all 12 clones were infected with garlic rust, although showing different reaction to the fungus. In two­year mean comparison, it was found that the observation of rust symptoms began from the second half of April, and disease progressed along with the growth of the plants. At the first stage Table 7 ­ Mean comparison of physiological traits in some Iranian garlic at two years In each column different letters mean significant differences between samples Table 8 ­ Mean comparison of physiological traits in some Iranian garlic at two years Treatment Leaf surface temperature °C Sub stomatal CO2 Stomatal conductance of H2O Photo­ synthesis Transpiration Chlorophyl Leaf dry weight Clove dry weight Gilvan 1 37.98 a 236.33 a 0.15 b 8.80 b 5.96 a 49.45 a 13.33 a 28.83 c Gilvan 2 36.65 ab 224.75 a 0.20 ab 13.16 a 6.08 a 58.53 a 13.42 a 29.44 bc Azar shahr 35.53 c 231.83 a 0.23 a 13.35 a 6.37 a 55.76 a 12.17 a 28.11 c Hesar 36.61 bc 233.50 a 0.22 a 12.85 a 6.46 a 57.26 a 13.05 a 28.66 c Lahijan 37.03 ab 249.50 a 0.21 ab 10.18 ab 6.31 a 58.70 a 12.38 a 31.92 a Khor va biabanak 37.30 ab 236.50 a 0.19 ab 11.47 ab 6.29 a 58.96 a 12.55 a 29.39 bc Sojas 36.98 ab 252 a 0.21 ab 10.78 ab 6.43 a 56.78 a 13.35 a 30.86 ab Dezfol 35.68 c 247.48 a 0.23 a 12.29 ab 6.28 a 55.45 a 12.32 a 28.76 c Sahneh 36.60 bc 250 a 0.23 a 12.27 ab 6.61 a 55.26 a 12.60 a 29.62 bc Khaf 37.65 ab 243.83 a 0.21 ab 11.50 ab 6.74 a 54.23 a 13.91 a 28.75 c Chavarzagh 37.28 ab 232.25 a 0.22 a 12.36 ab 6.80 a 55.13 a 12.43 a 31.29 a Lalejin 37.25 ab 238.08 a 0.19 ab 12.35 ab 6.15 a 55.68 a 12.40 a 28.76 c Treatment Nitrogen Phosphorus Potassium Manganese Zinc Copper Gilvan 1 4.73 ab 0.32 bcd 4.52 ab 42.53 a 24.61 c 8.06 bc Gilvan 2 4.60 ab 0.35 bc 4.47 abc 37.81 abc 28.69 bc 8.72 ab Azar shahr 4.67 abc 0.35 bc 4.30 bcd 40.58 ab 31.04 ab 8.52 abc Hesar 4.22 e 0.35 bc 3.84 ed 36.22 abc 24.80 c 8.71 ab Lahijan 4.76 ab 0.38 b 4.83 a 35.11 bc 29.03 bc 8.31 abc Khor va biabanak 4.66 abc 0.35 bc 4.01 cde 40.27 ab 29.48 bc 7.84 abc Sojas 4.47 cd 0.30 d 3.64 e 36.69 abc 26.63 bc 7.70 bc Dezfol 4.83 a 0.35 bc 4.85 a 42.77 a 26.98 bc 8.55 abc Sahneh 4.33 de 0.38 b 3.84 de 37.55 abc 28.53 bc 8.00 bc Khaf 4.32 de 0.35 bc 3.62 e 36.79 abc 26.71 bc 7.55 c Chavarzagh 4.77 ab 0.35 bc 4.51 abc 39.83 ab 26.74 bc 8.49 abc Lalejin 4.48 cd 0.44 a 3.62 e 30.89 c 35.78 a 9.21 a In each column different letters mean significant differences between samples Anjomshoaa et al. ‐ Iranian garlic resistance to rust fungus 549 of growth, the highest percentage of infection was related to the clone ‘Lahijan’. At the second stage, the highest percentage of infection was related to the clone ‘Khor va Biabanak and the lowest was relat­ ed to the clone ‘Hesar’. At the third and fourth stages, the clones ‘Gilvan1’ and ‘Lalejin’ had the low­ est percentage of infection and were identified as resistant to rust. This may indicate that garlic clones behave in different way during seedling and adult plant stages and therefore they have different genet­ ics for resistance to the garlic rust fungus in different growth stages. The most susceptible clone was ‘Chavarzagh’ showing the highest percentage of infection (Table 9). The first uredospores of garlic rust appeared in the first half of April each year, and the first teliospores appeared two weeks later. The onset and spread of infection completely follow the progressive growth pattern and with the help of ure dospores and in the absence of fungicides, the level of plant infection can reach from over 1% to more than 80% within two months. The results of this study showed that garlic clones respond differently to the rust and are separated into resistant, semi resistant, semi­susceptible and sus­ ceptible clones (Table 10). In sum, combined mean comparison of garlic clones in terms of resistance to disease showed that Gilvan1 cultivar had distinct traits compared to other clones. Morphologically, to color pf this clone is violet with code 5, 6­8 skins, side view shape of the compound bulb is broadly ovate, basal plate event, shape of matured bulb is globe and type of bulb structure is regular with two­fan group of cloves. The distinction of this clone with other vio­ let clones is in the number of skins. The next resistant clone, according to the field conditions of the present research, was ‘Lalejin’ from Hamadan Province, which has white stripes bulb with code 4 and white clove with code 1. The number of skins is 5­8 and side view shape of the compound bulb is broadly ovate, basal plate event, shape of matured bulb is broadly oval, and type of bulb structure is regular multi­shelled. Table 9 ­ Mean comparison of resistance to rust in some Iranian garlic clones in two year (Difference in days to maturity) Treatment Percentage of the disease 20/04/2019 05/05/2019 20/05/2019 20/06/2019 Gilvan 1 1.72 b 2.53 bc 3.97 c 5.83 c Gilvan 2 1.58 b 2.59 bc 4.65 abc 6.42 abc Azar shahr 1.72 b 2.99 abc 4.66 abc 6.30 bc Hesar 1.72 b 2.36 c 4.21 abc 6.25 bc Lahijan 2.01 a 2.86 abc 5.04 a 6.57 abc Khor va biabanak 1.72 b 3.32 a 4.92 ab 6.52 abc Sojas 1.72 b 2.45 bc 4.00 bc 5.97 bc Dezfol 1.72 b 3.00 abc 4.94 a 6.67 ab Sahneh 1.72 b 2.85 abc 4.29 abc 6.04 bc Khaf 1.72 b 3.03 ab 4.24 abc 5.95 bc Chavarzagh 1.72 b 2.88 abc 4.80 abc 7.08 a Lalejin 1.72 b 2.71 abc 4.55 abc 5.85 c In each column different letters mean significant differences between samples. Table 10 ­ Reaction of 12 Iranian garlic clones to rust fungus in a two years field traits under natural infection Clones Grouping of resistance Resistant Semi resistant Semi susceptible Susceptible Gilvan 1 x Lalejin x Khaf x Sojas x Sahneh x Hesar x Azar shahr x Gilvan 2 x Khor va biabanak x Lahijan x Dezfol x Chavarzagh x Adv. Hort. Sci., 2019 33(4): 543­552 550 Traits’ correlation The correlation coefficients between physiological traits and resistance to rust are shown in Table 11. The results of the study showed a positive and signifi­ cant correlation between the leaf surface tempera­ ture, photosynthesis, nitrogen and manganese uptake and percentage of leaf infection at the proba­ bility level of 1%. The relationship between the leaf surface temperature and infection frequency indi­ cates that by increasing the leaf temperature, the percentage of infection will be increased. This result is reasonable according to other researchers’ reports that the percentage of rust damage has only a direct correlation with the mean temperature. In addition, a positive and significant correlation was observed between percentage of rust infection and photosyn­ thesis and nitrogen, manganese and zinc. Also, a neg­ ative and significant correlation was found between percentage of rust infection and transpiration rate at the level of 1%. The correlation coefficients between morphologi­ cal traits and resistance to rust are shown in Table 12. A negative and significant correlation was observed between traits such as transpiration rate, bulb weight, bulb diameter, number of bulb skins and type of bulb structure and rust infection percentage at the 1% level. This relationship indicates that garlic rust is effective on the factors affecting the garlic yield, such as the bulb weight, bulb diameter and by increasing the percentage of infection, the value of these traits are reduced. Results of the infection frequency in four growth stages (Fig. 1) showed that the clones with high infec­ tion frequency at the early stages of growth had a higher disease symptomps at the final stages of growth. Therefore, the presence of infection at the early stages reduced the growth, yield and quality of the product. However, those that were resistant to some extent at the early stages of growth had rela­ tively low infection and slow spread of disease, and with better growth of the clones. 4. Discussion and Conclusions This is the first study on the identification of resis­ tant clones to rust on garlic through the study of morphological and physiological traits collected on Treat Percentage of the disease Percentage of the disease 1 Bulb weight ­0.462 ** Bulb diameter ­0.285 ** Mean clove weight 0.068 Number of bulb skin ­0.404 ** Number of cloves per bulb ­0.007 Leaf number 0.128 Color bulb 0.183 Color clove 0.009 Color leaf 0.096 Side view of bulb ­0.118 Shape of mature bulb ­0.098 Type of bulb structure ­0.526 ** Table 12 ­ Correlation coefficients between Morphological traits and resistance to rust (Puccinia allii) in Iranian garlic clones measured by Pearson's two­domain test Table 11­ Correlation coefficients between physiological traits and resistance to rust (Puccinia allii) in Iranian garlic clones measured by Pearson's two­domain test *, **, NS= significant at 5%, 1% and not significant probability levels, respectively. Treat Percentage of the disease Percentage of the disease 1 Leaf surface temperature ­ 0.602 ** Sub stomatal CO2 ­ 0.206 Stomatal conductance of H2O 0.059 Photosynthesis 0.411 ** Transpiration ­ 0.520 ** Chlorophyl 0.217 * Leaf dry weight 0.059 Clove dry weight 0.160 Nitrogen 0.625 ** Phosphorus ­ 0.085 Potassium 0.142 Manganese 0.298 ** Zinc 0.469 ** Copper 0.19 *, **, NS= significant at 5%, 1% and not significant probability levels, respectively. Fig. 1 ­ Correlation coefficients between Morphological traits and resistance to rust (Puccinia allii) in Iranian garlic clo­ nes measured by Pearson's two­domain test Anjomshoaa et al. ‐ Iranian garlic resistance to rust fungus 551 12 Iranian clones from Iran during two crop years. The clones ‘Gilvan1’ and ‘Lalejin’ had the lowest number of stomata in the leaf area, because the fun­ gus of the garlic rust through the stomata comes in to the plant, the low number of stomata reduces the possibility of pathogen entry and may be the most important feature of resistance in these clones. The results of two­year combined analysis of vari­ ance of morphological traits are consistent with the research report of Nourbakhshian et al. (2007). The results of their research showed that a significant dif­ ference was found between the number of cloves in the bulb, clove weight, length and diameter of the clove and amount of dry matter per unit area (Nourbakhshian et al., 2007). Vafaee and colleagues (2009), in a study on the genetic diversity of Iranian garlic clones using mor­ phological traits and AFLP as molecular markers, clas­ sified Iranian garlic clones in 6 main groups, while according to morphological traits, the total clones were classified into 4 general groups. The results of this study showed that garlic clones respond differently to the rust. The results of a study on resistance to garlic rust in Pakistan showed that rust severity of 0.8% was recorded on variety Hazro (Alam et al., 2007). A study in the US state of California regarding the susceptibility of the tested clones to garlic rust showed that none of the cultivars including selective later and early clones, and Spanish and Chinese cultivars showed complete resistance and were usually tolerant (Coviello, 2007). Another California breeding program to test the resistance to garlic rust on three genotypes PE493096, PE540315, W12820 showed that all of them were infected with garlic rust and only about 1% of them had less than 26% of the infection (Davis, 2007). The results of correlation coefficients between morphological traits and resistance to rust are consis­ tent with Nourbakhshian’s report on factors affecting garlic yield and bulb weight (Nourbakhshian et al., 2008). The results of this study are consistent with the results of other researchers regarding the superi­ or clones’ traits (Baghalian et al., 2005). Kallo report­ ed that a clove weight, number of cloves and bulb diameter had the most direct effect on garlic yield, and stated that the selection of the clones based on these traits would improve the yield potential of gar­ lic (Kallo, 1988). The use of commercial clones carrying rust resis­ tance genes is the most efficient, economical and environmental friendly method of rust disease con­ trol. Using morphological trait for early screening of the clones for resistance to garlic rust can be very important. The results of the infection frequency showed that the clones ‘Gilvan1’ and ‘Lalejin’ had the lowest percentage of infection and were identified as resistant clones to the rust. We found resistant clones with the introduction of each morphological trait. It should be noted that since the intensity measurements of infection were carried out at four stages, it was found that the earli­ est symptoms of garlic rust began in early May, and then the growing trend was accompanied by the growth of garlic. Two years of field trails lead us to achieve resistance components and to find resistant clones of garlic to the rust pathogen, however in order to get more in detail on mechanism and genet­ ics of resistance we are following genetics studies on resistance and susceptible clones. By this research we will find the diversity of genes involved in resis­ tance to rust pathogen in garlic and we will explore some more details on mechanisms of resistance in the future. References ALAM S.S., AHMAD M., USMAN A., AHMAD A., NAVEEDUL­ LAH A., 2007 ­ Variation in Garlic varieties for reaction to natural infection of ‘Puccinia porii’ and Alternaria porri. ­ Sarhad J. Agric., 23(1): 149­152. 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