ASHS 2024 Conference

A Look at Heat Stress on Micro-propagated Pecan Trees - Doris Alexa Arnedo
Unraveling the role of LEA(Late embryogenesis abundant) genes in pecan stress resilience - Sahithi Pulicherla
Discovery of Early Biomarkers for the Scab Resistance of Pecan Seedlings Using Metabolomic Analysis Combined with Machine Learning Algorithms - Min Jeong Kang
Developmental Transcriptomics of Pecan Fruit in ‘Mahan’ and ‘Tiny Tim’ - June Labbancz
Pistachio Orchard Productivity Enhancement Through Molecular Marker Preselection - Ewelina Jacygrad
Fatal Flaws of Experimental Almond Varieties and Selections - Luke Milliron
Advancing Cocoa Yield Forecasting in Ecuador Using Machine Learning and Field Data Integration - Daniel Mancero
Maturity stage at harvest modulates fruit softening and quality of jackfruit - Zora Singh
Growing papaya in Mississippi - Guihong Bi

Carya illinoinensis (pecan) belongs to the Juglandaceae family, and the native region extends from Illinois, USA to Oaxaca, Mexico. Pecan is a valuable economic crop due to its nutritious and tasty nuts, and the United States produced 275 million pounds of pecans in 2022. As temperatures are increasing it is important to understand the impact on pecan trees. By 2100, the average U.S. temperature is projected to increase by about 3°F to 12°F. Plants deal with heat stress in several different ways including the production of heat shock proteins (HSPs) and their transcription factors known as heat shock factors (HSFs). HSFs initiate the transcription of genes that encode heat shock proteins (HSPs), that deal with heat stress by initiating protein folding and aid in the repair or removal of damaged proteins. In this study, we aim to look at the genetic networks that are impacted when multiple genotypes are subjected to high-heat environments. For this study, seedstocks from multiple genotypes that span the geographic region of North America were introduced into micropropagation. These include seedstocks from ‘Elliott’, ‘Apache’, ‘Cape Fear’, ‘Mahan’, ‘Giles’, ‘Sioux’, ‘Wichita’, ‘Western’, and native seedstocks of unknown genetics from Ohio. A preliminary heat stress assay was performed on a micropropagated ‘Elliott’ line by subjecting three small trees to 43°C for two hours prior to flash freezing in liquid nitrogen and comparing these to the same clonal line (three trees) that remained at 23°C. Real time quantitative reverse transcription PCR (QRT-PCR) was performed on the heat stress and control trees. Normalized gene expression indicated that CiHSP1 expression was 2X higher in the heat-treated pecan trees than CiHSP1 expression of the control trees. The additional micropropagated seedstocks listed above are being subjected to heat stress at different temperature ranges and time intervals. The replicated assays will be analyzed using RNA-Seq and qRT-PCR to determine differential gene expression of control and heat-treated trees especially between the HSPs and HSFs. These assays will help determine the gene networks that pecan trees use as they experience heat stress and will help determine how different pecan genetics that originate in different geographic regions react to heat stress.

Late embryogenesis abundant (LEA) proteins, encoded by a family of LEA genes, are vital in conferring stress tolerance in plants through their unique intrinsically disordered structure that can stabilise cellular components under desiccated conditions. While the protective capabilities of LEA proteins are well-documented across various crops, their specific roles in pecan (Carya illinoinensis), a highly nutritious and economically significant nut crop, remain largely unexplored. This gap of knowledge needs to be addressed as pecan yields face threats from escalating drought and salinity issues, intensified by ongoing climate change. This study represents the first comprehensive analysis of LEA genes within the pecan genome. We have successfully identified 332 LEA genes distributed across 15 of the 16 chromosomes in four genomes of pecan, categorized into 8 distinct subgroups based on their conserved motif regions. Synteny analysis provided a deeper understanding of their evolutionary trajectories. Utilizing extensive transcriptomic datasets, we explored the tissue-specific expression patterns of LEA genes in pecan, discovering diverse expression profiles across various tissues. Ongoing studies include promoter analysis and assessments of gene expression under abiotic stress conditions. To specifically address the impact of drought, heat and salinity, clonal pecan plants are being subjected to these stressors under controlled conditions in tissue culture and greenhouse settings. This approach aims to directly observe the physiological and molecular responses of LEA genes under realistic stress simulations. The presence of LEA genes across a vast majority of pecan chromosomes and their diverse subgroup classifications suggests a genome-wide defense mechanism potentially key to enhancing the stress tolerance of pecan trees. By understanding and harnessing these genes, our research seeks to elucidate plant stress responses at the molecular level allowing the development of genetic strategies to ensure the sustainability of pecan by mitigating adverse environmental impacts on its production. This knowledge could also be applied in a diverse array of other economically significant crops.

Pecans (Carya illinoinensis (Wangenh.) K. Koch) are globally consumed nuts and an important agricultural commodity in the United States. Scab is a devastating pecan disease, which necessitates the application of numerous fungicide sprays in the growing season of pecans. Even with the control measures, in wet years, scab infection results in great yield loss (over 50% loss in susceptible varieties) and deterioration of nut quality. Although there have been various efforts to alleviate the scab, the development of scab-resistant pecan cultivars is the most effective method to control the disease. However, current methods to assess pecan scab resistance require multiple years of field screening and complicated laboratory (microscopic) techniques. Thus, a simple and reliable method that can rapidly evaluate pecan scab resistance at an early stage of infection is necessary. In this study, metabolomic analysis with machine learning algorithms was utilized to identify early biomarkers for the scab resistance of pecan seedlings. Two pecan seedlings with contrasting scab resistance ('Pawnee' and 'Desirable') were inoculated with water (control), Pa-OK-11 (isolated from 'Pawnee'), and De-Tif-11 (isolated from 'Desirable') for 7 days. 'Desirable' seedlings exhibited resistance to Pa-OK-11, while 'Pawnee' seedlings showed moderate resistance to De-Tif-11. Both cultivars were susceptible to their respective isolates. Leave samples from each seedling were collected at different time points (0, 1, 2, 3, 4, 5, 7 days). For the metabolomics work, liquid chromatography‒mass spectrometry (LC‒MS) was employed to analyze metabolites in samples, which can cover a wide range of primary and secondary metabolisms, including carbon fixation, glycolysis, citric acid cycle, amino acid metabolism, phenylpropanoid, monolignol, and flavonoid biosynthesis. Different machine learning algorithms were compared to find differentially regulated metabolites (biomarkers) between scab-resistant and -susceptible seedling groups. With a combination of machine learning models, we obtained reliable potential biomarkers, e.g., phenolic acids, flavonoids, plant hormones, and their intermediates and precursors, involved in the early stage of scab infection. The selected markers are expected to be used to classify scab resistance levels in pecan seedlings within a week after infection, which may replace the conventional method (phenotype-based mass selection) for pecan breeding selection. In short, this research breaks the bottleneck of resistance screening in pecans and will help facilitate the early selection of scab-resistant pecan cultivars to achieve breeding goals.

Pecan (Carya illinoinensis) is a nut crop native to the United States and Mexico which is becoming an increasingly important crop globally. Juglandaceous nuts are uniquely high in antioxidants among nuts and a conversion equivalent derived from studies in mice indicates that consumption of 22-38 pecans per day may reverse metabolic disorder in an individual weighing 132 pounds, implying a role in a healthy diet. Despite this importance, relatively little is known about the molecular basis of pecan nut ontogeny compared to other nut crops, leading to difficulties in understanding the physiological issues which plague growers. Susceptibility to various biotic and abiotic disorders including pecan scab, vivipary, water split, and shuck decline are dependent upon the stage of development the pecan nut is in. To better understand the molecular basis and timing of pecan nut development, developmental time-course RNA-Seq was carried out on nuts collected from cultivars ‘Mahan’ (a large nut bearing pecan from Mississippi) and ‘Tiny Tim’ (a small nut bearing native pecan from Missouri) approximately biweekly through the growing season of 2022. Using this data, genes were grouped together into distinct developmental phases, connecting transcriptional changes to the already well-characterized ontogenic stages of pecan nut development.

There is significant variation in tree size, which determines productivity, in commercial pistachio orchards planted with UCB-1 seedling rootstocks. It has been unclear to extent to which this is due to genetic differences or environmental variation. Nurseries have tried to tackle this problem by rogueing young seedlings before they are planted in orchards. However, our data previously demonstrated that performance in the first year is a poor predictor of later tree size. Genotyping by sequencing data from experimental and commercial orchards and genome wide association studies (GWAS), combined with our chromosome-scale, high quality, genome assemblies for the parental Pistacia atlantica and P. integerrima trees resulted in two highly informative molecular markers for vigor. Based on the genomic sequence information, we developed an inexpensive, quick, and easy qPCR protocol for single nucleotide polymorphism (SNP) marker analysis. We were able to predict the improved size distribution that extant orchards would have had if this marker had been used to rogue seedlings prior to planting in the orchards. We want this marker to make it available for nurseries to rogue out trees which would exhibit low vigor and productivity later in an orchard.

The California almond industry has funded multiple, multi-site almond variety evaluation trials over the last several decades. These field trials have previously evaluated many of the varieties that are now the most widely planted in California. Although, field evaluation trials are helpful for revealing which varieties are promising, they are, perhaps, most valuable to the industry for revealing which varieties/selection have serious flaws and should not be planted by growers. Thirty named cultivars and numbered breeder selections were planted in three replicated commercial orchards across California’s Central Valley in 2014. Of these 30, as of April 2024, one numbered selection Y116-161-99 from the USDA has been commercially released as ‘Yorizane’. However, nine of the 30 varieties/selections were dropped from further evaluation in the trial in 2022 for a variety of reasons: low yield (five), lack of interest by the breeder (two), extremely early bloom timing (one), and poor harvestability (one). Of the 21 still being evaluated in 2024, many have one of these major flaws, or additional flaws, that will likely prevent commercial adoption, including a high percentage of double kernels, susceptibility to bacterial blast (Pseudomonas syringae pv. syringae), Botryosphaeria canker disease susceptibility, hull rot susceptibility, and a high percentage of kernel creases or twins, just to name some of the additional flaws. Even if a variety/selection has high yield, good kernel quality, and none of these major flaws documented after ten years of evaluation (e.g. Y117-91-03 from USDA), further observation in the UC trial sites or in the orchards of early adopters may reveal important flaws that prevent sustained and widespread variety adoption. This long-term challenge is why some believe it takes decades to prove a new scion variety. The wide diversity of potentially fatal flaws underscores the need for cultivar evaluation to take place by a third-party like UC Cooperative Extension in long-term replicated trials to reduce substantial financial risk to the grower to the greatest extent possible. Keywords: Prunus dulcis, almond, variety evaluation, breeding, nut crop

This workshop will explore topics related to climate change impacts on temperate tree nut crops, including drought, salinity, freeze issues, and other environmental stressors. Additionally, participants will be given the opportunity to discuss relevance of topics to their specific commodity and share insights and suggestions with fellow researchers.

In this workshop we will be bringing in experts in pistachio and pecan cultivation to share their cutting-edge research and insights into mitigating the challenges posed by climate change. These esteemed speakers, who have dedicated their careers to understanding and addressing these issues, will provide valuable insight to workshop attendees and jumpstart discussion on this topic within groups. Attendees will have the opportunity to discuss relevant issues of climate change on their crop within small groups before bringing ideas to the entire group.

The impact of climate change on tree nut crops is likely to be significant, given expected decreases in chilling, increased droughts, and fluctuations in winter temperatures leading to decreases in production if mitigation measures are not implemented.

In this workshop, we seek to foster fruitful discussion among researchers working with tree nuts and other orchard crops likely to be impacted by climate change in order to brainstorm solutions and targets for future research in this topic area.

Coordinator(s)

• Lu Zhang, Oklahoma State University, Stillwater, OK, United States
• David Hlubik, Rutgers University, United States
• Phoebe Gordon, University of California Cooperative Extension, Madera, CA 93638, Madera County, United States

Speaker/Participant(s)

• Louise Ferguson, University of California Davis, Extension Specialist, Davis, California, United States
  Climate Impact on Tree Nut Crops - Pistachio Salinity (30 mins)
  Summary: Dr. Louise Ferguson is an Extension Specialist at University of California Davis with several decades of experience in pistachio and other orchard crops and will be speaking on pistachio salinity.
• Richard Heerema, New Mexico State University, Extension Specialist, Las Cruces, New Mexico, United States
  Climate Impact on Tree Nut Crops - Drought in Pecan (30 mins)
  Summary: Dr. Richard Heerema is an Extension Specialist at New Mexico State University with a focus on pecans and will be speaking on drought in pecan.
• Xinwang Wang, USDA ARS, College Station, TX, United States
  Climate Impact on Tree Nut Crops - Pecan Breeding Associated with Climate Change (30 mins)
  Summary: Dr. Xinwang Wang is a Research Geneticist for USDA ARS in College Station, TX and will be discussing challenges in pecan breeding associated with climate change.

Composted Pecan Shells: A Potential Growing Media Amendment For Container Grown Pecan Seedlings In Georgia - Srijana Thapa Magar
Oil and Dormex® improve bloom and yield in pistachios by driving metabolite changes - GURREET BRAR
Water requirements of Peacan Orchards in the Southeast US - Kriti Poudel

Pecan (Carya illinoinensis) production in Georgia holds significant economic importance nationally. It is an energy-intensive practice with a very low output-to-input ratio. Pecan byproducts, notably pecan shells and husks, account for up to 49% of the nut but are underutilized. A greenhouse experiment was conducted at the USDA facility in Byron, Georgia in 2023 to study the feasibility of composted pecan shells as a growing media amendment for container-grown pecan seedlings. The composted pecan shell was collected from a local pecan grower’s three-year-old composted pile, while fresh goat manure was sourced from the Fort Valley State University’s farm. Various ratios (25, 50, 75, and 100%) of composted pecan shells, along with biochar, goat manure, and chicken manure, were compared to a commercial soil mix (control). All the growing amendments underwent steam sterilization at 98°F for a couple of hours to eliminate any potential contaminants such as weeds, bacteria, fungi, and parasites. Each treatment combination was placed in individual floats to sow the one-year-old stratified ‘Elliott’ seeds. Once the seedlings developed two juvenile leaves, they were transferred to 3-gallon pots to evaluate further soil and plant physiological parameters. The treatments were arranged in a randomized complete block design with four blocks, each containing one treatment combination. Various soil and plant parameters were evaluated monthly, including soil electrical conductivity and temperature, plant size, photosynthesis, stem water potential, and chlorophyll content, to assess the impact of soil amendments on soil and pecan seedling growth. Results determined that composted pecan shell outperformed others in terms of germination (~80%), while none of the seeds germinated in any chicken manure treatment combination. Remarkably, the growth performance of pecan seedlings under different pecan shell ratios was comparable to those grown in commercial soil mix, biochar, and goat manure, indicating good plant health. The stem water potential values overall ranged above -6 Bar, suggesting no signs of plant water stress throughout the study. However, the 100% goat manure treatment consistently showed seedlings with significantly lower chlorophyll content and photosynthetic activity, leading to the smallest plant size compared to the control and biochar treatments. These findings highlight the potential of composted pecan shells as a sustainable soil amendment for container-grown pecan seedlings, offering a novel approach to repurpose pecan byproducts to enhance soil quality, promote sustainable agriculture practices, and serve as an additional income source to pecan growers, thus contributing to the economic viability of pecan production in Georgia.

Lack of dormant chilling is a major problem in producing pistachio trees in locations with warm periods during the winter time. In the past years, some locations in California have received insufficient winter chilling which has led to late bloom and crop reduction. Horticultural oil has been used as a rest-breaking agent to promote bud break and improve production. However, there is limited information regarding the merit of chill portion spray timing and the physiological mechanism behind bloom advancement by oil application. In the present study, three locations in California, North (Colusa County), Central (Madera County) and South-Western Fresno County (Cantua Creek) were selected for oil spray applications while Hydrogen cyanamide (Dormex®) was sprayed at Cantua Creek site. Tree of cv. Kerman (female) and Peters (male) on UCB-1 rootstock were sprayed with horticultural oil (IAP 440) @ 6% v/v or Dormex @4% at various chill portion (CP) accumulation milestones. Bloom period from bud swell to full bloom, tree yield, yield components, non-structural carbohydrates and macro and micro nutrients in buds and bark of pistachio shoots were analyzed. NMR-based metabolomics analysis was conducted to investigate the changes in metabolic profiles induced by exogenous oil or Dormex® application. Results showed that oil spraying in two southern locations advanced bud break but not in the northern site showing each location respond to oil spray differently. In Cantua site, Dormex® and oil spray at CP55 could significantly increase the yield while in Madera, oil spray at CP59 showed the highest yield. Results also showed that oil spray at different CPs and Dormex® could change the trend of soluble sugars and starch in bark and bud of pistachio trees. In Cantua, Dormex® significantly increased nitrogen (N), phosphorous (P), sulfur (S), boron (B), copper (Cu) and zinc (Zn) mobilization towards bud swell. Moreover, oil spray increased N, P, S in all CPs at all locations. A multivariate analysis conducted to compare the metabolite changes in control samples of bark and bud with these two rest-breaking agents led to the identification of nine metabolites that show a significant change in at least one of the comparisons (Creatine, Aspartate, Sucrose, Asparagine, Succinate, Fumarate, Leucine, Adenosine, and Uridine). It seems that oil and Dormex® applications can significantly increase the yield of pistachio trees by advancing bud break, improving bloom synchrony and also, by changes in carbohydrate, nutrients and metabolite changes in bark and bud of pistachio tree.

The state of Georgia is one of the states leading in pecan production in the U.S. However, the seminal papers on water management using state-of-art techniques like eddy-covariance system and micro lysimeters are far and few. Much of the information available on water-use efficiency arises from earlier studies done in the Southwest. However, crop varieties, Georgia soils, and rainfall, when coupled with a long, hot and humid climate of the Southeast, lead to contrasting water management practices from that of the Southwest. The present paper addresses the water needs of pecans in trees six to ten-year old throughout different physiological stages of the orchard in Georgia using the current irrigation schedule. Several record-breaking temperatures and the shifting rainfall patterns have had an important impact on pecan production. This precipitation variability further exacerbates the needs to tailor water-use efficiency to these new normal conditions. Along with the in situ meteorological data and soil moisture information, an eddy-covariance system is installed in Hawkinsville, Georgia. This poster shows how southeastern pecan orchards have different water requirements for optimum yield. This paper focuses on the differences between the current schedule, irrigation application at the farm and water-use data from the present study. Such results obtained likely for the first time will help to devise water management practices, optimize irrigation scheduling leading to increased water conservation and yield.
Keywords: water-use efficiency, production, climate change