Tomatillo (Physalis ixocarpa Brot. ex Horm.) is found among the horticulture crops destined for domestic consumption and with major importance in Mexico, it is only surpassed by chile (Capsicum annum L.), potato (Solanum tuberosum L.), tomato (Solanum lycopersicum L.) and onion (Allium cepa L.). The domestic area dedicated to this crop is 41,318 ha, with an average yield of 18.9 t ha-1 (Sistema de Información Agroalimentaria y Pesquera [SIAP], 2018), although diverse investigations mention that the potential of the best selected seeds can reach up to 45 t ha-1 (Peña-Lomelí, Ponce-Valerio, Sánchez-del Castillo, & Magaña-Lira, 2014). In this regard, there are perspectives to improve their importance by increasing yield and the per capita consumption (Santiaguillo-Hernández et al., 2012), this would increase the producers’ financial income by improving their productivity, industrialization and exportation.
Tomatillo is included in several regional dishes highly consumed in Mexico (Santiaguillo-Hernández, Vargas-Ponce, Grimaldo-Juárez, Sánchez-Martínez, & Magaña-Lira, 2009). Its use dates back to the Maya civilization and more recently to the Aztecs, where it was an integral part of their diet along with maize (Zea mays L.), beans (Phaseolus vulgaris L.) and chile. The species Physalis ixocarpa is native to Mexico and it is still found in the wild from Central America to the Unites States of America (Valtierra & Ramos, 2003).
The Autonomous University Chapingo has 407 Physalis accessions (sheltered in the National Bank of Vegetable Germplasm), which are a representative sample of the genetic variability found in Mexico (Santiaguillo-Hernández et al., 2012). It is important to evaluate the performance in the field of the germplasm sheltered ex situ, since it is a potential source of resistance genes to pathogens (Peña-Lomelí, Santiaguillo-Hernández, & Magaña-Lira, 2007). The accessions identified as tolerant or resistant could be incorporated to plant breeding programs to develop new commercial varieties (Pérez-Grajales, Márquez-Sánchez, & Peña-Lomelí, 1998).
Diseases are one of the main agents that reduce crop yield, therefore it is necessary to know the causal agent in order to generate efficient control strategies that will avoid aggravating the sanitary state of the crop. In order to do so, accurate diagnosis obtained from substantial basic information is required (Apodaca-Sánchez, Barreras-Soto, Cortez-Mondaca, & Quintero-Benítez, 2008).
Tomatillo, as most of the cultivated plants, is attacked by a diversity of pathogens among which fungi and viruses stand out. Some fungi have been identified and their pathology is known. However, there is still the need to study a lot of pathogens with financial significance in this crop. Such is the case of the organism that causes the disease known as “white smut” or “frog-eye”, which has not been satisfactorily controlled because it reduces yield considerably when it interacts with other pathogens or attacks by itself. This disease occurs in successive crop cycles which increases its occurrence and therefore its damage. This requires the design of a satisfactory control through a thorough study of the causal agent (Piña-Aguilar & Ponce-González, 1990).
Based on the aforementioned information, the objective of this research was to evaluate the damage caused by Entyloma australe Speg. (white smut) in 24 tomatillo materials (Physalis ixocarpa Brot. ex Horm.) with the purpose of determining their degree of resistance or susceptibility, as well as the impact of this pathogen in fruit yield.
Materials and methods
The study was made in the agricultural cycles spring-summer and summer-fall in 2017. In both cases, it was established in Plot X-3 in the Experimental Agricultural Field of the Autonomous University Chapingo, Mexico; located in the geographical coordinates 19° 29’ 20.4’’ north latitude and 98° 52’ 26.7’’ west longitude, at 2,250 masl. This plot was selected because in previous studies, in the spring-summer cycle in 2014, 2015 and 2016, tomatillo cultivations were attacked by white smut, thus it was considered a plot infested with this disease.
The prevailing climate in Chapingo is Cb(Wo)(W)b(i’), defined as temperate sub-humid with rains in the summer, with less than 5 % rainfall in the winter, slight thermal oscillation, fresh summers and an annual average rainfall of 636 mm. The mean annual temperature in this area is between 12 and 18 °C, with a variation of less than 5 °C (García, 1988).
Seventeen accessions of Physalis spp. pertaining to the Germplasm Bank of Native Species of the Crop Science Department of the Autonomous University Chapingo (Table 1) were evaluated to determine if some could be resistant to the attack of Entyloma australe Speg. These accessions were selected based on a previous study (unpublished information), in which they were listed as candidate accessions resistant to the tobacco mosaic virus, cucumber mosaic virus, alfalfa mosaic virus, tomato spotted wilt virus and the tabaco etch virus. Additionally, seven varieties from the plant breeding program of tomatillo of the Autonomous University Chapingo were evaluated (Table 1).
|Accession number||Code||Accession number||Code|
|5||82 BNGEN||66||Verde 01|
|11||173 BNGEN||87||181 BNGEN|
|12||JAL 103||106||209 BNGEN|
|14||184 BNGEN||133||JAL 01|
|15||128 BNGEN||172||167 BNGEN|
|26||48 BNGEN||204||Puebla SM31|
|35||170 BNGEN||202||Zafiro MSM1|
|55||148 BNGEN||201||Manzano T1|
|57||156 BNGEN||205||Tecozautla 041|
In the spring-summer cycle in 2017, the accessions were sowed on February 25, commercial substrate peat moss was used as well as 200 cavity polystyrene trays. Two to three seeds were placed per cavity and subsequently one plant was left per cavity. Seedlings were kept under greenhouse conditions and they were irrigated every three days with a Steiner nutrient solution (Steiner, 1984) at 50 % for three weeks; afterwards, they were irrigated on a daily basis with a Steiner nutrient solution at 100 %. The field was prepared with one fallow and one crossed-harrowing, spacing between furrows was of 1.2 m. Each furrow was prepared with a 16 mm pipe with self-compensated droppers and with a distance of 30 cm between them. Transplanting was carried out 34 days after sowing.
A randomized blocks design with four repetitions was established, placing 22 plants per experimental unit in a furrow of 6.3 m in length and 1.2 m in width, with a distance of 30 cm between plants. Deep-fertilization was applied with commercial products urea, diammonium phosphate and potassium chloride (100 N - 100 P2O5 - 50 K2O, respectively). During the development of the crop, 50 kg of urea per hectare were irrigated each week. Nutrition was completed with applications of foliar liquid fertilizer (Bayfolan Forte® + adhesive) to provide minor elements. Plagues were controlled with Imidacloprid (Confidor®) and Methomyl 90 % (Lannate®), and weed control was carried out by hand hoeing and hand weeding.
In the summer-fall cycle in 2017, sowing was carried out on June 24. Seedbed and land preparation management was the same as the previous cycle, transplanting was carried out 41 days after sowing at a distance of 30 cm between plants. In this case, 15 plants were placed per experimental unit. Repetitions, plant distribution and the experimental design were the same as the previous cycle, as well as fertilizations, plague and weed control.
The characteristics evaluated in the first cycle were:
Occurrence. Determined by the number of sick plants per experimental unit 15 days after transplanting (dat), every 15 days in five samplings.
Severity. In this variable, the intensity of the symptoms in the plants was considered by using the Doble Digit scale proposed by Saari and Prescott (1975) and modified by Eyal, Scharen, Prescott and van Ginkel (1987). The Doble Digit scale specifies the location or height of the disease in a plant through the first digit and the second digit indicates the level or percentage of damage (1 = 0 %, 2 = 25 %, 3 = 50 %, 4 = 75 % y 5 = 100 %). Severity evaluations were carried out every 15 dat. A total of five evaluations were made.
Area under the disease progress curve (AUDPC). The occurrence and severity data obtained was used to determine the progress of the disease and the response of the materials to the fungus, through the calculation of the AUDPC analysis, which was calculated with the trapeizodal rule integration method (Campbell & Madden, 1990) using the following equation:
where n is the number of measurements of the disease in time, (Y i+1 +Y i )/2 is the midpoint between Y i y Y i+1 which represents the amount of the disease in a time interval corresponding to the height of each rectangle, and X i+1 -X i represents the amount of time (d) between two evaluations of the disease and it indicates the width of the trapezoid.
Plant height (cm). It was measured 30 dat in 10 plants previously tagged, from the surface of the ground to the tip of the highest branch.
Yield per plant. This variable was determined in the first (RP1, kg per plant) and second (RP2; kg per plant) harvest carried out 72 and 83 dat respectively, and total yield per plant (TYP) was obtained by adding RP1 and RP2.
Weight (g). the weight of 10 fruits was determined in the first (PF1) and second (PF2) harvest, the average weight (AFW) of the fruits was obtained afterwards from the information gathered in the first and second harvest.
Volume of fruits of the first harvest (VOL; cm3). This variable was estimated through the volume of water displaced by the fruits in a 2 L graduated cylinder containing 1 L of water.
In the second harvest cycle it was not possible to evaluate all of the aforementioned variables because in addition to the damage caused by Entyloma australe Speg., there were severe symptoms caused by Fusarium spp., which prevented the plant’s development. In the second harvest, only PH, occurrence, severity and AUDPC were evaluated and registered the same way as in the first cycle, but in this cycle only four samplings were carried out.
In order to carry out the morphological characterization of the pathogen, sick vegetable material was collected. Then, a cluster sampling was carried out (Cochran, 1977) in a cinco de oros pattern. In each cluster, from a symptomatic leaf per plant, Koch’s postulates were tested to confirm the presence of the pathogen. Finally, it was compared with the morphology described by Fischer (1953), Holliday (1980) and Zundel (1953).
The effect of each evaluated population on the variables was studied through a variance analysis and Tukey’s comparison test (P ≤ 0.05). The statistical analyses were made with SAS program version 9.3 (SAS Institute Inc., 2011).
Results and discussion
During the first cultivating cycle, symptomatology was observed 30 dat, through yellowish brown spots more or less circular and slightly lifted on the backside of the leaf, resembling blisters. These characteristics coincide with the description made by Romero (1988). With Koch’s postulates, it was confirmed that the symptoms corresponded to Entyloma australe Speg.
In the second cycle, the disease became obvious 15 dat with the same symptomatology described by Romero (1988). Due to intensive rain and the difficulty to weed periodically, as it is usual in this crop, plants did not receive proper nutrition, which had a possible influence on its susceptibility (Velasco-Velasco, 1999). In addition to the aforementioned information, this cycle also presented Fusarium spp., according to the symptomatology described by Agrios (2005), which caused the death of many plants.
The results of the variance analysis of the first cycle (spring-summer 2017) show a highly significant effect of the populations on the nine evaluated and analyzed variables (Table 2), which was also observed in the second cultivating cycle (summer-fall 2017) in PH and AUDPC (Table 3).
|Source of variation||DF||PH||AUDPC|
|Coefficient of variation||17.1||87.9|
Comparison of means tests of the first cultivating cycle
The results in the comparison of means test of the first cultivating cycle (Table 4) show that the variety Manzano T was the highest (erect), although it was statistically the same as the varieties Puebla SM3 and Gema, and accessions 5 (82 BNGEN), 15 (128 BNGEN), 39 (Verde L.), 57 (156 BNGEN) and 172 (167 BNGEN). Accession 12 (JAL 103) showed the lowest PH, but it was the same as (P ≤ 0.05) the other eleven accessions, which means that their growth habit is creeping or prostrate. Peña-Lomelí et al. (2011) describe that there is genetic diversity in growth habits in tomatillo that vary from erect, semi-erect and prostrate, which coincide with what was found in the behavior of the evaluated materials.
|5 (82 BNGEN)||80.05 abcz||27.1 ij||132.9 ab||160.0 h-k|
|11 (173 BNGEN)||74.75 bcd||191.5 f-j||129.2 ab||320.7 d-k|
|12 (JAL 103)||58.75 e||550.6 a-d||89.3 b||639.9 a-d|
|14 (184 BNGEN)||75.7 bcd||386.9 c-f||168.3 ab||555.2 b-f|
|15 (128 BNGEN)||79.25 a-d||168.8 f-j||214.0 ab||382.8 c-j|
|18 (32 BNGEN)||73.625 b-e||105.3 hij||123.3 ab||228.5 g-k|
|26 (48 BNGEN)||73.2 b-e||7.7 j||59.4 b||67.1 jk|
|35 (170 BNGEN)||71.125 b-e||260.3 efgh||205.3 ab||465.6 b-h|
|39 (Verde L.)||77.125 a-d||23.6 ij||103.7 ab||127.3 ijk|
|55 (148 BNGEN)||74.075 b-e||316.5 e-h||167.9 ab||484.3 b-g|
|57 (156 BNGEN)||83.85 ab||118.2 g-j||130.6 ab||248.8 f-k|
|64 (147 BNGEN)||63.725 de||319.3 d-h||84.7 b||404.0 c-i|
|66 (Verde 01)||75.75 bcd||255.2 e-i||137.3 ab||392.5 c-i|
|87 (181 BNGEN)||65.725 cde||220.0 f-j||56.6 b||276.6 f-k|
|106 (209 BNGEN)||74.125 b-e||212.9 f-j||101.4 b||314.4 e-k|
|133 (JAL 01)||69.475 b-e||11.4 j||44.8 b||56.2 k|
|172 (167 BNGEN)||82.7 ab||188.6 f-j||144.8 ab||333.3 d-k|
|203 (Gabriela)||69.05 b-e||563.3 abc||161.7 ab||725.0 ab|
|204 (Puebla SM3)||80.325 abc||349.6 c-g||305.9 a||655.5 abc|
|202 (Zafiro MSM)||69.225 b-e||455.2 b-e||156.3 ab||611.5 a-e|
|200 (Diamante)||74.95 bcd||764.8 a||121.0 ab||885.8 a|
|201 (Manzano T)||92.425 a||754.9 a||129.2 ab||884.1 a|
|205 (Tecozautla 04)||76.125 bcd||674.5 ab||229.1 ab||903.7 a|
|31 (Gema)||78.45 a-d||669.7 ab||238.4 ab||908.1 a|
Yield and size of the fruit
In the results obtained for the variables regarding yield (Table 4), it can be observed that the control varieties (Diamante, Manzano T, Zafiro MSM, Gabriela, Puebla SM3, Tecozautla 04 y Gema) were superior to the accessions of the germplasm bank, except 12 (JAL 103). This is because the control evaluated varieties are product of the plant breeding program (Sánchez-Martínez & Peña-Lomelí, 2015), where the main selection criteria has been yield, and the accessions are cultivated as well as wild accessions (Peña-Lomelí et al., 2008, 2014).
The variety Tecozautla 04 was the highest in average fruit weight and volume (biggest fruit size), but it was the same as (P ≤ 0.05) Gema, Manzano T and Puebla SM3 in AFW and as Gema, Manzano T, Puebla SM3, Diamante and Gabriela in VOL (Table 5). This indicated that the varieties Diamante and Gabriela have big fruits, but less specific weight, this could be because the breeding program has focused in the number and size of the fruits as main selection criteria (Peña-Lomelí et al., 2002; Santiaguillo-Hernández, Cervantes-Santana, & Peña-Lomelí, 2004).
Regarding the evaluated accessions, 12 (JAL 103) presented the highest fruit yield. The accessions that had the lowest yields and smaller fruit size were 26 (48 BNGEN) and 133 (JAL 01) because they are wild materials and of less productive potential (Peña-Lomelí & Santiaguillo-Hernández, 1999; Peña-Lomelí et al., 2011)
|5 (82 BNGEN)||17.5 fz||30.8 jk||53.8 hi||42.3 gh||629.6 a-d|
|11 (173 BNGEN)||102.5 f||111.0 g-k||93.0 ghi||102.0 fgh||166.7 ef|
|12 (JAL 103)||362.5 b-e||377.0 def||163.5 e-h||270.3 de||729.0 ab|
|14 (184 BNGEN)||187.5 def||211.3 f-j||124.0 ghi||167.6 efg||188.5 ef|
|15 (128 BNGEN)||171.3 def||189.8 g-k||151.3 e-i||170.5 ef||265.5 c-f|
|18 (32 BNGEN)||87.5 f||104.8 h-k||82.0 ghi||93.4 fgh||245.7 c-f|
|26 (48 BNGEN)||13.5 f||24.3 k||20.3 i||21.6 h||136.2 ef|
|35 (170 BNGEN)||225.0 c-f||253.0 fgh||130.3 ghi||191.6 ef||393.8 a-f|
|39 (Verde L.)||31.3 f||43.8 ijk||38.8 hi||41.3 gh||197.4 ef|
|55 (148 BNGEN)||203.8 c-f||223.5 f-i||163.8 e-h||193.6 ef||153.0 ef|
|57 (156 BNGEN)||110.0 f||150.8 g-k||115.8 ghi||133.3 fgh||354.3 b-f|
|64 (147 BNGEN)||368.8 b-e||385.3 c-f||190.3 d-g||287.8 de||56.3 f|
|66 (Verde 01)||150.0 ef||186.8 g-k||153.0 e-i||169.9 ef||262.5 c-f|
|87 (181 BNGEN)||222.5 c-f||291.0 efg||145.8 f-i||184.8 ef||249.7 c-f|
|106 (209 BNGEN)||193.8 def||228.3 fgh||172.5 d-h||200.4 ef||447.9 a-f|
|133 (JAL 01)||13.8 f||20.8 k||24.0 i||22.4 h||748.0 ab|
|172 (167 BNGEN)||133.8 ef||135.0 g-k||116.0 ghi||125.5 fgh||212.2 ef|
|203 (Gabriela)||443.8 abc||444.3 cde||276.8 c-f||360.5 cd||393.8 a-f|
|204 (Puebla SM3)||527.5 ab||547.8 bcd||444.5 a||496.1 ab||652.1 abc|
|202 (Zafiro MSM)||233.8 c-f||240.8 fgh||155.3 e-i||198.0 ef||150.0 ef|
|200 (Diamante)||418.8 a-d||565.0 abc||283.8 cde||424.4 bc||225.0 def|
|201 (Manzano T)||662.5 a||647.5 ab||305.8 bcd||476.6 abc||305.4 c-f|
|205 (Tecozautla 04)||662.5 a||686.0 ab||436.3 ab||561.1 a||799.7 a|
|31 (Gema)||627.5 a||734.3 a||364.5 abc||549.4 ab||503.6 a-e|
Area under the disease progress curve
Regarding AUDPC in the first cultivating cycle, accessions 64 (147 BNGEN), 26 (48 BNGEN), 11 (173 BNGEN), 14 (184 BNGEN), 39 (Verde L), 55 (148 BNGEN) and 172 (167 BNGEN) stood out due to their major degree of tolerance to Entyloma australe Speg. (Table 5), but not due to their yield and fruit size (Table 4 and 5), which coincide with Escalante-Ortiz and Farrera-Pino (2004), who indicate that lower AUDPC values correspond to materials with less disease severity. It is worth highlighting that in previous unpublished research, accessions 26 (48 BNGEN), 12 (JAL 103), 15 (128 BNGEN), 56 (88 BNGEN), 64 (147 BNGEN) and 147 (201 BNGEN) showed resistance to Fusarium oxysporum in an independent evaluation. Based on the aforementioned information, it can be mentioned that accessions 26 (48 BNGEN) and 64 (BNGEN) are of high value to a plant breeding program for disease resistance (Peña-Lomelí et al., 2007). Within the group of improved varieties Zafiro MSM and Diamante stood out because they were the least affected by the disease (Table 5).
Comparison of means tests of the second cultivating cycle
In the evaluation of the second cultivating cycle (Table 6), accession 18 (32 BNGEN) and the Puebla SM3 variety showed the highest PH, whilst accession 133 (JAL 01) showed the least PH. In this evaluation cycle, heights were more homogeneous compared to the evaluation in the first cycle (Table 4), because the development period of the crop took place during the rainy season, which caused the development of the disease under study as well as other pathogens such as Fusarium spp. This situation prevented the full expression of the evaluated populations’ agronomic behavior.
|5 (82 BNGEN)||15 abcz||325 bcd|
|11 (173 BNGEN)||14 bc||400 bcd|
|12 (JAL 103)||15 abc||900 a|
|14 (184 BNGEN)||15 abc||250 cd|
|15 (128 BNGEN)||12 bc||200 cd|
|18 (32 BNGEN)||21 a||400 bcd|
|26 (48 BNGEN)||14 abc||125 cd|
|35 (170 BNGEN)||14 bc||300 bcd|
|39 (Verde L.)||16 abc||175 cd|
|55 (148 BNGEN)||17 abc||325 bcd|
|57 (156 BNGEN)||16 abc||475 bc|
|64 (147 BNGEN)||14 abc||350 bcd|
|66 (Verde 01)||16 abc||75 cd|
|87 (181 BNGEN)||14 abc||400 bcd|
|106 (209 BNGEN)||12 bc||400 bcd|
|133 (JAL 01)||11 c||50 d|
|172 (167 BNGEN)||12 bc||225 cd|
|203 (Población 3)||15 abc||425 bcd|
|204 (Puebla SM3)||21 a||425 bcd|
|202 (Zafiro MSM)||12 bc||75 cd|
|200 (Diamante)||15 abc||250 cd|
|201 (Manzano T)||18 ab||4 75 bc|
|205 (Tecozautla 04)||18 ab||675 ab|
|31 (Gema)||16 abc||475 bc|
Area under the disease progress curve
In this variable, accession 133 (JAL 103) showed major tolerance to the attack of Entyloma australe Speg., and accession 12 (JAL 103) the highest level of susceptibility (Table 6). In the case of the registered varieties, only Diamante and Zafiro MSM showed certain level of tolerance to the fungus. The two accessions with major resistance in the first cycle were 26 (48 BNGEN) and 64 (BNGEN) and in the second one they were not different (P ≥ 0.05) from 133 (JAL 01), which indicates that these varieties are of high value as possible resistance sources to Entyloma. The latter could be due to the synthesis of secondary metabolites produced by the plant for its defense (Vivanco, Cosio, Loyola-Vargas, & Flores, 2005).
Accession 133 (JAL 01) is a wild accession, thus it can only be used as a resistance source to the disease. However, its behavior was not consistent in both evaluation cycles, possibly because the first one favored the severity of the fungus, therefore this interaction with the environment modified the degree of tolerance (Agrios, 2005). The key to successful plant breeding is to have continuous provision of genetic variability and of favorable characters contained within this diversity (Dwivedi et al., 2008). Until now, genes of wild plants have provided cultivars with resistance to pests (Malik, Brown-Guedira, Smith, Harvey, & Gill, 2003) and diseases (Brar, 2005), that is why accession 133 is of high value to a plant breeding program for resistance to Entyloma australe Speg.
Due to the behavior of Diamante and Zafiro MSM, both of these varieties are candidates to incorporate disease resistance by crossbreeding them with the identified wild populations.
Accessions 26 (48 BNGEN) and 64 (147 BNGEN) were identified as potential sources of resistance genes to Entyloma australe Speg. because they were more tolerant to the attack of this pathogen; although their yield was not the highest.
The evaluated accessions present different yield potential, which is not associated to its resistance to the attack of Entyloma australe Speg., given that accession 12 (JAL 103) obtained higher yield, even though it was the most susceptible to the attack of the disease.
The cultivated varieties with less susceptibility were Zafiro MSM and Diamante, and even though they did not show the highest yield and their fruit size was intermediate, they were less affected by the attack of the fungus. In contrast, the varieties Tecozautla 04, Manzano T and Gema showed the highest yield and fruit size, but they were also the most susceptible materials.