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This research was carried out from 2004 to 2005 in two commercial ‘Hass’ avocado orchards cultivated under rainfed conditions in a hot subhumid climate of the state of Nayarit, Mexico. The objectives of this study were to: 1) establish the patterns in nutrient concentrations during the lifespan of winter and summer vegetative flush leaves; and 2) validate a methodology based on mathematical functions to identify the appropriate period for leaf sampling to diagnose plant nutrition in avocado considering its two major vegetative flushes. Leaf samples were taken monthly for each vegetative flush, starting when leaf length was 5 cm or greater and concluding at leaf abscission. Starting at vegetative budbreak, winter and summer leaves lived 12.5 and 7.8 months, respectively. Summer flush leaves grew faster and attained greater length than winter leaves. A mathematical model based on the concentration of macroand micronutrients through the lifespan of avocado leaves was evaluated. This model was used to determine the period when nutrient concentrations became stable and, consequently, to identify the proper leaf sampling period. For the ‘Hass’ avocado in Nayarit, the period for sampling winter flush leaves corresponded to 6.6to 7.9-month-old leaves (4 Sept. to 13 Oct.). For summer leaves the optimum period was shorter and occurred when leaves were 3.9 to 4.9 months old (5 Dec. to 5 Jan.). The procedure and sampling time obtained here should be tested in other regions. Leaf analysis is an important tool for proper nutrition management in avocado orchards. It serves for diagnosis of deficiencies, excesses, or nutritional imbalances. It is also useful for making fertilizer recommendations and monitoring the response of trees to fertilizer applications (Salazar-Garcı́a, 2002). In most commercial avocado orchards, 4to 7-month-old leaves are sampled for nutrient analyses (Crowley et al., 1993; Dixon et al., 2007; Maldonado-Torres et al., 2007; Salazar-Garcı́a, 2002). Two criteria have been established to sample avocado leaves for nutrient analyses: 1) leaves must be fully expanded; and 2) nutrient concentrations must be stable. The second criterion is usually met by determining nutrient concentrations and visually detecting the stabilization period. The disadvantage of using the visual criterion is not having an exact date for the beginning and end of the stabilization period, because only data corresponding to each of the sampling dates are known. Besides this, estimating nutrient concentration in the leaves between sampling dates is not exact; therefore, determining the period of stabilization is subjective. An additional problem is that the units used to present the various nutrient concentrations on a graph are different (i.e., % or mg·kg); therefore, the variations in performance of all the nutrients on one single graph cannot be accurately appreciated. In the state of Nayarit, ‘Hass’ avocado is cultivated without irrigation (rainfed) and produces two vegetative flushes. The winter flush occurs in January to February and is the most important because it produces the largest proportion of floral buds in next year’s flowering; in addition, it supports fruit development until maturity. The summer flush occurs during the rainy season (July to August) and is of intermediate importance for flowering (Cossio-Vargas et al., 2008). In Nayarit, ‘Hass’ avocado presents only one flowering flush during the winter and a single harvest period in October to December of the same year (Salazar-Garcı́a et al., 2007a). The lifespan of avocado leaves may differ depending on the vegetative flush of origin and the demand for nutrients and other compounds in critical phases of tree phenology as well as stresses that cause or accelerate their abscission (Liu et al., 1999; Roets et al., 2006). A 10to 12-month life expectancy is mentioned for ‘Hass’ avocado leaves (Whiley and Schaffer, 1994), although in Nayarit, winter and summer flush ‘Hass’ leaves live 12 and 9 months, respectively (Salazar-Garcı́a et al., 2007b). This research was undertaken to determine or validate the leaf sampling period currently used for ‘Hass’ avocado in Nayarit and had the following objectives: 1) to establish the patterns in nutrient concentrations during the lifetime of winter and summer vegetative flush leaves; and 2) to validate a methodology to identify the appropriate leaf sampling period for nutritional diagnosis considering its two major vegetative flushes. Materials and Methods Plant material. Two rainfed commercial orchards of ‘Hass’ avocado grafted to local West Indian seedling rootstocks were studied in 2004–05. Orchards were on a Chromic Cambisol medium texture soil (INEGI, 1999) in the hot subhumid climate of the municipality of Tepic, Nayarit (Garcı́a-Amaro, 1998). The ‘‘Nájera’’ orchard was located in La Yerba (lat. 21 31.6# N, long. 105 02.9# W) at 858 m above sea level (asl). The ‘‘Ante’’ orchard was situated in Venustiano Carranza (lat. 21 32.0# N, long. 104 59.1# W) at 927 m asl. In both orchards average mean annual rainfall was 1225 mm, which occurred from June to September. At the beginning of the study, trees were 10 years old and established at a spacing of 8 · 8 m. Orchards received growers’ standard management practices using a site-specific fertilization program (Salazar-Garcı́a et al., 2009; Salazar-Garcı́a and Lazcano-Ferrat, 2003). The fertilization program was slightly modified every year according to yield, soil, and leaf analysis. However, in the 2 years before this study, the following nutrient amounts per tree were applied to the soil each year: 2.14 kg nitrogen (N) (ammonium sulphate 20.5% N and urea 46% N), 0.324 kg phosphorus (P) (diammonium phosphate 18% N, 46% P2O5), 2.10 kg potassium (K) (potassium sulphate 50% K2O), 0.061 kg magnesium (Mg) (K-MAG 22% K2O, 18% MgO, 22% SO4), 0.810 kg zinc (Zn) (zinc sulphate 36% Zn), and 30.2 g boron (B) (Boronat 32% B2O3). Except N, fertilizers were applied by hand in solid form around each tree in a 20-cm-deep · 30-cm-wide trench at 1.5 to 2 m away from the trunk. Nitrogen was applied by hand to the soil canopy shadow area and split in three applications (July, August, and September). All the P and Mg were applied in July; the rest of nutrients were split in two applications (July and September). Received for publication 29 Aug. 2014. Accepted for publication 27 Oct. 2014. This investigation was partially funded by INIFAP, the Consejo Nacional de Ciencia y Tecnologı́a (CONACYT), the FOMIXNayarit, and the Fundación Produce Nayarit. We express our appreciation to Alberto Ante and Juan Nájera for allowing the use of their avocado orchards as well as Luis E. Cossio-Vargas and José González-Valdivia for their technical support. Special thanks to Dr. Ricardo Goenaga for reading the manuscript. Independent Researcher. To whom reprint requests should be addressed; e-mail samuelsalazar@prodigy.net.mx. 130 HORTSCIENCE VOL. 50(1) JANUARY 2015 Soil sampling. From each orchard, a mixed soil sample was obtained from five of the 10 trees previously selected at a 0to 30-cm depth. In Apr. 2005, two subsamples were taken from each tree with a stainless steel auger. A subsample was taken halfway between the tree’s trunk and the dripline zone, and the other one from the drip line zone. Texture, pH (1:2 H2O), electrical conductivity, organic matter, N-inorganic, P-Bray, K, calcium (Ca), Mg, sodium (Na), iron (Fe), Zn, copper (Cu), manganese (Mn), and B were determined in a commercial laboratory under The North American Proficiency Testing Program of the Soil Science Society of America. Leaf sampling. In each orchard, trees with historical yields 100 kg or greater were identified. From this population, 10 trees were randomly chosen and in each tree, a cohort of 300 winter and 300 summer proleptic vegetative shoots was tagged at the time of budbreak. This corresponded to the zero time for winter (19 Feb. 2004) and summer (11 Aug. 2004) vegetative shoots. From these dates onward, days after budbreak (DABB) were counted up to the date of leaf abscission. Leaf samples were collected monthly alternating between odd and even numbered trees (five trees per sampling date) and began when the leaves reached 5 cm or greater in length and finished at leaf abscission. At each sampling date, 20 healthy, fully expanded leaves (leaf blade + petiole), situated in basipetal positions 6 and 7, were collected per tree. A total of 12 leaf samples for winter (Apr. 2004 to Mar. 2005) and eight for summer (Sept. 2004 to Apr. 2005) vegetative flushes were collected within the first week of each month. At each sampling time, the length of 10 leaves (blade) per tree per orchard was measured. Leaves were washed and dehydrated in a Laboratory-line Imperial-5 digital forced-air oven (3488M USA) at 70 C, until constant weight; subsequently, the samples were ground in a Thomas Scientific (Wiley Mini Mill 3383-L10) stainless steel mill with a 40-caliber mesh and analyzed for N, P, K, Ca, Mg, sulfur (S), Fe, Cu, Mn, Zn, and B in a commercial laboratory accredited by The North American Proficiency Testing Program of the Soil Science Society of America. Appropriate leaf sampling period. Mathematical functions were generated using DABB as the independent variable and the concentrations of each nutrient as dependent variables. The general equation was: Nutrient 1⁄4 b0 þ b1Dþ b2D þ b3D þ b4D þ b5D; where D = days after budbreak and b = mathematical coefficients Afterward, the best mathematical function according to its order of response (second to fifth order) for each nutrient was selected using the Stepwise procedure (SAS, 2009). The criteria for choosing the best functions were: 1) value of R; 2) a small mean square error; and 3) the value of Cp (Draper and Smith, 1981; Neter et al., 1985). Once the best mathematical functions were identified, their mathematical coefficients (b0, ... bn) were calculated by the REG procedure, according to the afor

Identification of the Appropriate Leaf Sampling Period for Nutrient Analysis in ‘Hass’ Avocado