Transcriptome profile of pecan scab resistant and susceptible trees from a pecan provenance collection.

Pecan scab is a devastating disease that causes damage to pecan (Carya illinoinensis (Wangenh.) K. Koch) fruit and leaves. The disease is caused by the fungus Venturia effusa (G. Winter) and the main management practice for controlling the disease is by application of fungicides at 2-to-3-week intervals throughout the growing season. Besides disease-related yield loss, application of fungicides can result in considerable cost and increases the likelihood of fungicide resistance developing in the pathogen. Resistant cultivars are available for pecan growers; although, in several cases resistance has been overcome as the pathogen adapts to infect resistant hosts. Despite the importance of host resistance in scab management, there is little information regarding the molecular basis of genetic resistance to pecan scab.The purpose of this study was to elucidate mechanisms of natural pecan scab resistance by analyzing transcripts that are differentially expressed in pecan leaf samples from scab resistant and susceptible trees. The leaf samples were collected from trees in a provenance collection orchard that represents the natural range of pecan in the US and Mexico. Trees in the orchard have been exposed to natural scab infections since planting in 1989, and scab ratings were collected over three seasons. Based on this data, ten susceptible trees and ten resistant trees were selected for analysis. RNA-seq data was collected and analyzed for diseased and non-diseased parts of susceptible trees as well as for resistant trees. A total of 313 genes were found to be differentially expressed when comparing resistant and susceptible trees without disease. For susceptible samples showing scab symptoms, 1,454 genes were identified as differentially expressed compared to non-diseased susceptible samples. Many genes involved in pathogen recognition, defense responses, and signal transduction were up-regulated in diseased samples of susceptible trees, whereas differentially expressed genes in pecan scab resistant samples were generally down-regulated compared to non-diseased susceptible samples.Our results provide the first account of candidate genes involved in resistance/susceptibility to pecan scab under natural conditions in a pecan orchard. This information can be used to aid pecan breeding programs and development of biotechnology-based approaches for generating pecan cultivars with more durable scab resistance.

Enhancing entomopathogenic nematode efficacy with Pheromones: A field study targeting the pecan weevil.

Consistent efficacy is required for entomopathogenic nematodes to gain wider adoption as biocontrol agents. Recently, we demonstrated that when exposed to nematode pheromone blends, entomopathogenic nematodes showed increased dispersal, infectivity, and efficacy under laboratory and greenhouse conditions. Prior to this study, the impact of entomopathogenic nematode-pheromone combinations on field efficacy had yet to be studied. Steinernema feltiae is a commercially available entomopathogenic nematode that has been shown to increase mortality in insect pests such as the pecan weevil Curculio caryae. In this study, the pecan weevil was used as a model system to evaluate changes in S. feltiae efficacy when treated with a partially purified ascaroside pheromone blend. Following exposure to the pheromone blend, the efficacy of S. feltiae significantly increased as measured with decreased C. caryae survival despite unfavorable environmental conditions. The results of this study highlight a potential new avenue for using entomopathogenic nematodes in field conditions. With increased efficacy, using entomopathogenic nematodes will reduce reliance on conventional management methods in pecan production, translating into more environmentally acceptable practices.

Pecan (Carya illinoinensis (Wangenh.) K. Koch) nuts as an emerging source of protein: extraction, physicochemical and functional properties.

BACKGROUND: The increasing demand for sustainable alternatives to traditional protein sources, driven by population growth, underscores the importance of protein in a healthy diet. Pecan (Carya illinoinensis (Wangenh.) K. Koch) nuts are currently underutilized as plant-based proteins but hold great potential in the food industry. However, there is insufficient information available on pecan protein, particularly its protein fractions. This study aimed to explore the physicochemical and functional properties of protein isolate and the main protein fraction glutelin extracted from pecan nuts. RESULTS: The results revealed that glutelin (820.67 ± 69.42 g kg-1) had a higher crude protein content compared to the protein isolate (618.43 ± 27.35 g kg-1), while both proteins exhibited amino acid profiles sufficient for adult requirements. The isoelectric points of protein isolate and glutelin were determined to be pH 4.0 and pH 5.0, respectively. The denaturation temperature of the protein isolate (90.23 °C) was higher than that of glutelin (87.43 °C), indicating a more organized and stable conformation. This is further supported by the fact that the protein isolate had a more stable main secondary structure than glutelin. Both proteins demonstrated improved solubility, emulsifying, and foaming properties at pH levels deviating from their isoelectric points in U-shaped curves. Compared to the protein isolate, glutelin displayed superior water and oil absorption capacity along with enhanced gelling ability. CONCLUSION: The protein isolate and glutelin from pecan nuts exhibited improved stability and competitive functional properties, respectively. The appropriate utilization of these two proteins will support their potential as natural ingredients in various food systems. © 2024 Society of Chemical Industry.

Metabolomics based on GC-MS revealed hub metabolites of pecan seeds germinating at different temperatures.

BACKGROUND: As an important plant source of food and edible oils, pecans are rich in metabolites. Few studies have focused on metabolites involved in pecan seed germination at different temperatures. RESULTS: In our study, we germinated pecan seeds at different temperatures and found that, the germination rate and water content were highest at 30°C. It was found that the radicle of pecan seeds could sense seed coat cracking by observing the microstructure and cell ultra-structure of the seeds at the early stage of germination. We compared the metabolomes of seeds at different temperatures with different germination processes. A total of 349 metabolites were identified, including 138 primary metabolites and 211 secondary metabolites. KEGG enrichment analysis indicated that the differential metabolites were mainly enriched in the metabolic pathways, amino acid synthesis pathways and ABC transporters. Using weighted gene co-expression network analysis (WGCNA), three modules of closely related metabolites were identified. In the brown module, most of hub metabolites were amino substances, whereas in the blue module, many hub metabolites were sugars. CONCLUSIONS: Amino acids and carbohydrates play an important role in pecan seed germination. Differential metaboliteanalysis showed that 30°C was the temperature at which metabolites differed most significantly. This study provides useful information for further research on the seedling establishment of pecan seeds.

First report of Fusarium concentricum as a causal agent of Fusarium leaf blotch on pecan (Carya illinoinensis) in Southeast China.

The economically important nut crop pecan (Carya illinoinensis (Wangenh.) K. Koch) is seriously affected by increasing incidence of fungal disease worldwide (Xiao et al 2021). The top leaves of the pecan variety 'Pawnee' in the orchard of Zhejiang A&F University, Zhejiang, China were damaged by massive dark brown plaques in summer to autumn 2021. The causal agent was isolated from leaves with target plaques following the steps: sterilized with 70% alcohol (30 s × 2), rinsed with sterilized water (3 ×) before and after 5% sodium hypochlorite (30 s), excised the plaques, and placed on PDA medium at 28℃ in a dark incubator for 3-d. The mycelium on the edge of each colony was transferred to fresh SNA medium in dark for 2 weeks to induce conidia formation. A few conidia-germinated mycelia were transferredand inoculated on new plates containing fresh PDA medium to obtain the purified cultures. Koch's postulates were applied to validate the pathogenicity of the purified isolates. Non-woundedly healthy leaves (disinfected with 5% sodium hypochlorite) of 'Pawnee' seedlings were inoculated with 5 mm 7-d old purified cultures. Dark-brown spots appeared on the leaves 2 days post inoculation at 25℃. The spots became larger accompanied by partially cracking and slight deformation of inoculated leaves from day 2 to day 4, while the control leaves remained asymptomatic. A re-isolated strain ZJ-6 from these infected leaves was identified as the pathogenic isolate with the same symptom as the previous one. Morphologically, aerial mycelia of the pathogenic isolate ZJ-6 cashmere and white. The reverse of colony orange. The edge of the colony appeared gradually thinner, the aerial mycelia loose and flocky, and the matrix mycelium whitened. Hyphae were septate, translucent with smooth wall and 1.47-7.14 µm in width. Microconidia (n = 20) obovoid to fusoid, mainly with 0-septate, 4.45-7.78×4.79-16.25 µm. Macroconidia (n = 20) sickle, mainly with 3-5 septa, 5.56-10.28×56.67-114.54 µm. Simultaneous of monophialidic and polyphialidic conidiophores. Conidiophore width 1.47-3.68 µm, slightly smaller than vegetative hyphae. The morphological characteristics matched with previous descriptions of Fusarium species (Nirenberg and O'Donnell 1998; Wang et al 2013). The identity of ZJ-6 was confirmed by phylogenetic reconstruction using the concatenated sequences of the ATP citrate lyase (ACL1), Calmodulin (CaM), the internal transcribed spacer (ITS) rDNA region, ribosomal RNA gene (LSU), the largest subunit of DNA-dependent RNA polymerase II (RPB1), partial translation elongation factor-1 alpha (TEF) and ß-Tubulin (TUB). To this end, the genomic DNA of ZJ-6 was extracted by the M5 hipermix-MF859 (Mei5 Biotechnology) and submitted to GenBank under the accession numbers OP933646, OP933647, OP925890, OP925889, OP933396, OP933648, and OP933397, respectively. The obtained sequences of ZJ-6 were used for nucleotide BLAST against thetandard databases, respectively, and the strains with sequence identification values above 98% were selected to construct multiple alignment for building a phylogenetic tree. This analyses allowed the identification of ZJ-6 as Fusarium concentricum Nirenberg & O'Donnell, a species with few reports that can cause serious damage to the fruits and branches of other hosts (Hasan et al 2020; Huda-Shakirah et al 2020; Wang et al 2013). This is the first report of pathogenic F. concentricum on pecan in Southeast China that caused no harvest of infected plants.

First report of leaf spot on pecan caused by Choanephora cucurbitarum in China.

Pecan (Carya illinoinensis K. Koch) is an important and widely planted nut tree species in Jiangsu Province, China (Mo et al. 2018). In July 2020, leaf spot symptoms were frequently observed on pecan in Jurong, Jiangsu Province (119°15'36"E, 32°1'6"N). Disease incidences ranged from 40 to 65% among 150 mature pecan trees from three nurseries. The disease severity index (DSI, Jiang et al. 2019) reached 58.4. Symptoms began as small brown spots scattered on leaves that gradually expanded to large, circular to irregular black and brown necrotic lesions. In severe cases, lesions developed on large portions of a single leaf, and eventually the dead leaves fell from the trees. Three monoconidial isolates (Chen2346, Chen2347, Chen2348) were isolated from lesion margins and cultured on potato dextrose agar (PDA) medium. Colonies on PDA were white and cottony, later becoming light gray with abundant reproductive structures. Sporangiophores were aseptate, hyaline, unbranched, and apically dilated to form a clavate vesicle, which produced sporangia. Sporangia were globular to ellipsoid, brown to dark brown, 103 to 128 µm in length, and 88 to 114 µm in width (n = 30). Sporangiola were brown, ellipsoid to ovoid, with longitudinal striae, and measured 13.9 to 18.8 × 7.9 to 10.8 µm (n = 60). The morphological characteristics of these isolates agreed with descriptions of Choanephora cucurbitarum (Kirk 1984). Genomic DNA of these three monoconidial isolates was extracted, and the partial sequences of the internal transcribed spacer (ITS) and large subunit (LSU) of rDNA were amplified using primer pairs ITS1/ITS4 (White et al. 1990) and LR0R/LR7 (Vilgalys and Hester 1990), respectively. The consensus sequences (GenBank accession nos.: OP315248 to OP315250 for ITS and OP315251 to OP315253 for LSU) were aligned using BLASTn and showed100% identity with the sequences from C. cucurbitarum found in GenBank (accession nos.: MF942131 for ITS and ON025788 for LSU). To further confirm the identity, a phylogenetic analysis was performed with MEGA (v.7.0) and MrBayes (v.3.1.2) software, using the maximum likelihood and Bayesian inference methods, respectively. The multigene phylogeny revealed that the three isolates in this study, as well as, C. cucurbitarum specimen, clustered as a strongly supported monophyletic group (99 bootstrap value; 0.95 posterior probabilities). Based on the morphological and molecular data, the isolates were identified as C. cucurbitarum. To confirm pathogenicity, healthy pecan seedlings (2 years old) were each inoculated with a mycelial block (3 × 3 mm) excised from the margin of a colony on the vein of each leaf. Seedlings treated with non-colonized PDA blocks were used as controls. The inoculated seedlings were maintained in sterile plastic boxes with moistened sheets of filter paper at 25 ± 1°C and a 12-h photoperiod. Fifteen leaves per isolate were tested for each treatment. The experiment was repeated twice. Three days after inoculation, symptoms similar to those in the field appeared, whereas the control leaves remained symptomless. Subsequently, C. cucurbitarum was reisolated from the lesions and morphologically identified, confirming Koch's postulates. To the best of our knowledge, this is the first report of C. cucurbitarum causing leaf spot on C. illinoinensis in China. This study provides the foundation to further investigate the biology, epidemiology, and management of this disease.

Genome-Wide Identification, Characterization, and Expression Analysis of Long-Chain Acyl-CoA Synthetases in Carya illinoinensis under Different Treatments.

As crucial enzymes in the lipid metabolic network, long-chain acyl-CoA synthases (LACSs) are members of the acyl-activated enzyme superfamily and play a crucial role in epidermal wax synthesis, plant lipid anabolic metabolism, and stress tolerance. In this study, 11 pecan LACS genes were identified and categorized into five groups and located on nine chromosomes. The significant degree of conservation in the AtLACS and CiLACS protein sequences was demonstrated by multiple sequence alignment and conserved motif analysis. Cis-acting element analysis identified numerous stress-responsive and hormone-inducible elements in the promoter regions of CiLACS genes. The expression levels of CiLACS9 and CiLACS9-1 were considerably up-regulated under salt and drought stress, according to the qRT-RCR study. Treatment with ABA also led to increased expression levels of CiLACS1, CiLACS1-1, CiLACS2, and CiLACS9-1. Notably, CiLACS4, CiLACS4-1, CiLACS9, and CiLACS9-1 exhibited peak expression levels at 135 days after anthesis and are likely to have been crucial in the accumulation of seed kernel oil. Moreover, the CiLACS9 gene was shown to be located in the cytoplasm. These findings offer a theoretical framework for clarifying the roles of LACS genes in the processes of pecan kernel oil synthesis and response to abiotic stressors.

Multiple Mutations and Overexpression in the CYP51A and B Genes Lead to Decreased Sensitivity of Venturia effusa to Tebuconazole.

Multiple demethylation-inhibiting (DMI) fungicides are used to control pecan scab, caused by Venturia effusa. To compare the efficacy of various DMI fungicides on V. effusa, field trials were conducted at multiple locations applying fungicides to individual pecan terminals. In vitro assays were conducted to test the sensitivity of V. effusa isolates from multiple locations to various concentrations of tebuconazole. Both studies confirmed high levels of resistance to tebuconazole. To investigate the mechanism of resistance, two copies of the CYP51 gene, CYP51A and CYP51B, of resistant and sensitive isolates were sequenced and scanned for mutations. In the CYP51A gene, mutation at codon 444 (G444D), and in the CYP51B gene, mutations at codon 357 (G357H) and 177 (I77T/I77L) were found in resistant isolates. Expression analysis of CYP51A and CYP51B revealed enhanced expression in the resistant isolates compared to the sensitive isolates. There were 3.0- and 1.9-fold increases in gene expression in the resistant isolates compared to the sensitive isolates for the CYP51A and CYP51B genes, respectively. Therefore, two potential mechanisms-multiple point mutations and gene over expression in the CYP51 gene of V. effusa isolates-were revealed as likely reasons for the observed resistance in isolates of V. effusa to tebuconazole.

First Report of Scab Disease Caused by Venturia effusa on Pecan in Anhui Province of China.

Pecan (Carya illinoinensis) is a world-famous nut tree which widely cultivated in China. Quanjiao County, located in Anhui province, is reputed to be the capital of pecan production in China. Since 2019, typical scab symptoms were observed on most pecan cultivars in orchards located in the regions of Quanjiao (32°5'7.08″ N, 118°16'2.91″ E). In April, dark brown to black lesions of scab could be observed on both the abaxial and adaxial surface of the lamina, and were often associated with the veins or midrib. In July, small, brownish, and circular lesions ranging from 1 to 2 mm in diameter were observed at the end of stems and shoulder of the fruit. In the surveyed orchards, disease incidence on the leaves reached more than 35%. While, according to the number of infected nut clusters, disease incidence ranged from 40 to 60% on the infected fruits. Using a sterilized scalpel, conidia were scraped from the surface of a single lesion from the infected leaves or fruits, and a dilute spore suspension was prepared in sterile distilled water, of which 100 microliters was spread on 1% water-agar plate (Bock et al. 2014). The conidia were incubated at 25°C for 48 h under fluorescent lights with a 12-hphotoperiod. Single germinated conidia were selected and transferred into potato dextrose agar (PDA) plate to obtain monospore isolates. From 2019 to 2020, more than 20 isolates were obtained from the infected leaves and fruits. Incubated at 24°C for 6 weeks in darkness on PDA, the colonies were gray-black with circular morphology and floccose texture, which were consistent with the characteristics of Venturia effusa described previously (Gottwald 1982). The conidia were pyriform to ellipsoid, zero to one septate, smooth, attenuated towards apex and base, base truncate, pale brown and 10.08 to 18.14 × 4.86 to 9.56 µm (n = 50) in size. To further identify the isolates, the regions of internal transcribed spacer (ITS), beta-tubulin 2 (TUB2) and translation elongation factor 1 alpha (EF1-a) were amplified and sequenced from genomic DNA for the three representative isolates (AH-81 and AH-82 from the infected leaves, and AH-41 from the infected fruits), respectively (White et al. 1990; Young et al. 2018; Bensch et al. 2006). Sequences of them were deposited in GenBank under nos. OP199056 to OP199058 (ITS), OP566581 to OP566583 (TUB2) and OP566578 to OP566580 (EF1-a). Multilocus phylogenetic analysis revealed that three isolates and V. effusa were clustered in the same clade, indicating high genetic similarity between these organisms. Their morphological and molecular characteristics were consistent with those for V. effusa. The pathogenicity of three isolates were tested on two-year-old container-grown pecan seedlings, which were grown in the nursery. The conidial suspension with a concentration of 5 × 105 conidia/ml was sprayed evenly on the surface of leaves of a healthy pecan seedling, and each isolate inoculated four pecan seedlings. The pathogenicity experiment was repeated three times. The plants inoculated with sterile water were used a negative control. The inoculated plants were enclosed in plastic bags for 2 days, and kept in the nursery greenhouse. Four weeks after inoculation, a similar symptom of scab was observed on leaves of cultivar Mahan, and V. effusa was isolated again from inoculated leaves with the frequency of 100% by the single-spore isolation, whereas no symptoms were observed on the control plants. To our knowledge, this is the first report of V. effusa as a scab pathogen on pecan in Anhui Province of China and underscores the need for monitoring this disease and developing disease control strategies to prevent severe reduction in the value of fruit. References: Bensch, K., et al. 2006. Studies in Mycology, 55(1): 299-305. Bock, C. H., et al. 2014. Forest Pathology, 44(4): 266-275. Gottwald, T. R. 1982. Taxonomy of the pecan scab fungus Cladosporium caryigenum. Mycologia. 74 (3), 382-390. White, T. T., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Application. Academic Press, San Diego, CA. Young, C. A., et al. 2018. Phytopathology, 108(7): 837-846. The author(s) declare no conflict of interest. Keywords: Venturia effusa, Scab, Pecan, Identification †Indicates the corresponding author.Y. Q. Zhao; zhaoyuqiang123@126.com.

Genome-Wide Identification and Expression Analysis of AMT and NRT Gene Family in Pecan (Carya illinoinensis) Seedlings Revealed a Preference for NH4+-N.

Nitrogen (N) is a major limiting factor for plant growth and crop production. The use of N fertilizer in forestry production is increasing each year, but the loss is substantial. Mastering the regulatory mechanisms of N uptake and transport is a key way to improve plant nitrogen use efficiency (NUE). However, this has rarely been studied in pecans. In this study, 10 AMT and 69 NRT gene family members were identified and systematically analyzed from the whole pecan genome using a bioinformatics approach, and the expression patterns of AMT and NRT genes and the uptake characteristics of NH4+ and NO3- in pecan were analyzed by aeroponic cultivation at varying NH4+/NO3- ratios (0/0, 0/100,25/75, 50/50, 75/25,100/0 as CK, T1, T2, T3, T4, and T5). The results showed that gene duplication was the main reason for the amplification of the AMT and NRT gene families in pecan, both of which experienced purifying selection. Based on qRT-PCR results, CiAMTs were primarily expressed in roots, and CiNRTs were majorly expressed in leaves, which were consistent with the distribution of pecan NH4+ and NO3- concentrations in the organs. The expression levels of CiAMTs and CiNRTs were mainly significantly upregulated under N deficiency and T4 treatment. Meanwhile, T4 treatment significantly increased the NH4+, NO3-, and NO2- concentrations as well as the Vmax and Km values of NH4+ and NO3- in pecans, and Vmax/Km indicated that pecan seedlings preferred to absorb NH4+. In summary, considering the single N source of T5, we suggested that the NH4+/NO3- ratio of 75:25 was more beneficial to improve the NUE of pecan, thus increasing pecan yield, which provides a theoretical basis for promoting the scale development of pecan and provides a basis for further identification of the functions of AMT and NRT genes in the N uptake and transport process of pecan.

Identification and Expression Analysis of MPK and MKK Gene Families in Pecan (Carya illinoinensis).

Mitogen-activated protein kinases consist of three kinase modules composed of MPKs, MKKs, and MPKKKs. As members of the protein kinase (PK) superfamily, they are involved in various processes, such as developmental programs, cell division, hormonal progression, and signaling responses to biotic and abiotic stresses. In this study, a total of 18 MPKs and 10 MKKs were annotated on the pecan genome, all of which could be classified into four subgroups, respectively. The gene structures and conserved sequences of family members in the same branch were relatively similar. All MPK proteins had a conserved motif TxY, and D(L/I/V)K and VGTxxYMSPER existed in all MKK proteins. Duplication events contributed largely to the expansion of the pecan MPK and MKK gene families. Phylogenetic analysis of protein sequences from six plants indicated that species evolution occurred in pecan. Organ-specific expression profiles of MPK and MKK showed functional diversity. Ka/Ks values indicated that all genes with duplicated events underwent strong negative selection. Seven CiPawMPK and four CiPawMKK genes with high expression levels were screened by transcriptomic data from different organs, and these candidates were validated by qRT-PCR analysis of hormone-treated and stressed samples.

Genome-Wide Identification, Characterization, and Expression Profiling of AP2/ERF Superfamily Genes under Different Development and Abiotic Stress Conditions in Pecan (Carya illinoinensis).

The ethylene-responsive element (AP2/ERF) is one of the keys and conserved transcription factors (TFs) in plants that play a vital role in regulating plant growth, development, and stress response. A total of 202 AP2/ERF genes were identified from the pecan genome and renamed according to the chromosomal distribution of the CiAP2/ERF genes. They were divided into four subfamilies according to the domain and phylogenetic analysis, including 26 AP2, 168 ERF, six RAV, and two Soloist gene family members. These genes were distributed randomly across the 16 chromosomes, and we found 19 tandem and 146 segmental duplications which arose from ancient duplication events. The gene structure and conserved motif analysis demonstrated the conserved nature of intron/exon organization and motifs among the AP2/ERF genes. Several cis-regulatory elements, which were related to light responsiveness, stress, and defense responses, were identified in the promoter regions of AP2/ERFs. The expression profiling of 202 CiAP2/ERF genes was assessed by using RNA-Seq data and qRT-PCR during development (pistillate flowering development, graft union development, and kernel development) and under abiotic stresses (waterlogging, drought). Moreover, the results suggested that the ERF-VII members may play a critical role in waterlogging stress. These findings provided new insights into AP2/ERF gene evolution and divergence in pecan and can be considered a valuable resource for further functional validation, as well as for utilization in a stress-resistance-variety development program.

Transcriptome Analysis Reveals a Comprehensive Virus Resistance Response Mechanism in Pecan Infected by a Novel Badnavirus Pecan Virus.

Pecan leaf-variegated plant, which was infected with a novel badnavirus named pecan mosaic virus (PMV) detected by small RNA deep sequencing, is a vital model plant for studying the molecular mechanism of retaining green or chlorosis of virus-infected leaves. In this report, PMV infection in pecan leaves induced PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI). PMV infection suppressed the expressions of key genes of fatty acid, oleic acid (C18:1), and very-long-chain fatty acids (VLCFA) biosynthesis, indicating that fatty acids-derived signaling was one of the important defense pathways in response to PMV infection in pecan. PMV infection in pecans enhanced the expressions of pathogenesis-related protein 1 (PR1). However, the transcripts of phenylalanine ammonia-lyase (PAL) and isochorismate synthase (ICS) were downregulated, indicating that salicylic acid (SA) biosynthesis was blocked in pecan infected with PMV. Meanwhile, disruption of auxin signaling affected the activation of the jasmonic acid (JA) pathway. Thus, C18:1 and JA signals are involved in response to PMV infection in pecan. In PMV-infected yellow leaves, damaged chloroplast structure and activation of mitogen-activated protein kinase 3 (MPK3) inhibited photosynthesis. Cytokinin and SA biosynthesis was blocked, leading to plants losing immune responses and systemic acquired resistance (SAR). The repression of photosynthesis and the induction of sink metabolism in the infected tissue led to dramatic changes in carbohydrate partitioning. On the contrary, the green leaves of PMV infection in pecan plants had whole cell tissue structure and chloroplast clustering, establishing a strong antiviral immunity system. Cytokinin biosynthesis and signaling transductions were remarkably strengthened, activating plant immune responses. Meanwhile, cytokinin accumulation in green leaves induced partial SA biosynthesis and gained comparatively higher SAR compared to that of yellow leaves. Disturbance of the ribosome biogenesis might enhance the resistance to PMV infection in pecan and lead to leaves staying green.

Transcriptomic Analysis to Unravel Potential Pathways and Genes Involved in Pecan (Carya illinoinensis) Resistance to Pestalotiopsis microspora.

Fruit black spot (FBS), a fungal disease of pecan (Carya illinoinensis (Wangenh) K. Koch) caused by the pathogen Pestalotiopsis microspora, is a serious disease and poses a critical threat to pecan yield and quality. However, the details of pecan responses to FBS infection at the transcriptional level remain to be elucidated. In present study, we used RNA-Seq to analyze differential gene expression in three pecan cultivars with varied resistance to FBS infection: Xinxuan-4 (X4), Mahan (M), and Wichita (W), which were categorized as having low, mild, and high susceptibility to FBS, respectively. Nine RNA-Seq libraries were constructed, comprising a total of 58.56 Gb of high-quality bases, and 2420, 4380, and 8754 differentially expressed genes (DEGs) with |log2Fold change| ≥ 1 and p-value < 0.05 were identified between M vs. X4, W vs. M, and W vs. X4, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathway analyses were performed to further annotate DEGs that were part of specific pathways, which revealed that out of 134 total pathways, MAPK signaling pathway, plant−pathogen interaction, and plant hormone signal transduction were highly enriched. Transcriptomic profiling analysis revealed that 1681 pathogen-related genes (PRGs), including 24 genes encoding WRKY transcription factors, potentially participate in the process of defense against Pestalotiopsis microspora infection in pecan. The correlation of WRKY TFs and PRGs was also performed to reveal the potential interaction networks among disease-resistance/pathogenesis-related genes and WRKY TFs. Expression profiling of nine genes annotated as TIFY, WRKY TF, and disease-resistance protein-related genes was performed using qRT-PCR, and the results were correlated with RNA-Seq data. This study provides valuable information on the molecular basis of pecan−Pestalotiopsis microspora interaction mechanisms and offers a repertoire of candidate genes related to pecan fruit response to FBS infection.

Oxidative Stress Amelioration of Novel Peptides Extracted from Enzymatic Hydrolysates of Chinese Pecan Cake.

Pecan (Carya cathayensis) is an important economic crop, and its hydrolyzed peptides have been evidenced to reduce the effect of oxidative stress due to their antioxidant capacity. Hence, the protocols of ultrafiltration and gel filtration chromatography were established to obtain bioactive peptides from by-products of C. cathayensis (pecan cake). As measured by DPPH/ABTS radical scavenging, the peptides with less molecular weight (MW) possess higher antioxidant capacity. PCPH-III (MW < 3 kDa) presented higher radical scavenging capacity than PCPH-II (3 kDa < MW < 10 kDa) and PCPH-I (MW > 10 kDa) measured by DPPH (IC50: 111.0 µg/ mL) and measured by ABTs (IC50: 402.9 µg/mL). The secondary structure and amino acid composition varied by their MW, in which PCPH-II contained more α-helices (26.71%) and ß-sheets (36.96%), PCPH-III contained higher ratios of ß-turns (36.87%), while the composition of different secondary of PCPH-I was even 25 ± 5.76%. The variation trend of α-helix and random experienced slightly varied from PCPH-I to PCPH-II, while significantly decreased from PCPH-II to PCPH-III. The increasing antioxidant capacity is followed by the content of proline, and PCPH-III had the highest composition (8.03%). With regard to the six peptides identified by LC-MS/MS, two of them (VYGYADK and VLFSNY) showed stronger antioxidant capacity than others. In silico molecular docking demonstrated their combining abilities with a transcription factor Kelch-like ECH-associated protein 1 (Keap1) and speculated that they inhibit oxidative stress through activating the Keap1-Nrf2-ARE pathway. Meanwhile, increased activity of SOD and CAT­antioxidant markers­were found in H2O2-induced cells. The residue of tyrosine was demonstrated to contribute the most antioxidant capacity of VYGYADK and its position affected less. This study provided a novel peptide screening and by-product utilization process that can be applied in natural product developments.

Brazilian Agroindustrial Wastes as a Potential Resource of Bioative Compounds and Their Antimicrobial and Antioxidant Activities.

The study of the recovery of bioactive compounds from natural resources and its implications in several areas is very significant for the scientific community. This work aimed to study Brazilian agroindustrial wastes' antioxidant and antimicrobial activities using green extraction. Olive leaves, jaboticaba peel, araçá peel, and pecan nut shells were evaluated under four conditions: (1) convective-drying and aqueous extraction, (2) convective-drying and ethanolic extraction, (3) freeze-drying and aqueous extraction, and (4) freeze-drying and ethanolic extraction. The results demonstrated that all samples showed high antioxidant potential, and the highest antioxidant activity was obtained for the extract of pecan nut shell. As for the quantification of compounds by HPLC, the olive leaf presented the highest content of phenolic compounds in the extract, mainly oleuropein. Finally, the antimicrobial activity analysis revealed the extracts' bactericidal potential against Staphylococcus aureus and Escherichia coli. The present study shows that green extraction can extract bioactive compounds with antioxidant and antimicrobial properties, highlighting the importance of choosing the drying method and solvent for future uses of these natural resources by the industry.

Comparative Analysis of the Transcriptomes of Persisting and Abscised Fruitlets: Insights into Plant Hormone and Carbohydrate Metabolism Regulated Self-Thinning of Pecan Fruitlets during the Early Stage.

Pecan is one of the most popular nut species in the world. The fruit drop rate of the pecan 'Pawnee' is more than 57%, with four fruit drop stages, which is very serious. In this study, we conducted transcriptomic profiling of persisting and abscised fruitlets in early fruit development by RNA-seq. A total of 11,976 differentially expressed genes (DEGs) were identified, 3012 upregulated and 8964 downregulated, in a comparison of abscised vs. persisting fruitlets at 35 days after anthesis (DAA). Our transcriptomic data suggest that gene subsets encoding elements involving the biosynthesis, metabolism, perception, signal transduction, and crosstalk of the plant hormones abscisic acid (ABA), auxin, cytokinin, ethylene, and gibberellin (GA) and plant growth regulators jasmonates, salicylic acid, and brassinosteroids were differentially expressed. In addition, the majority of transcriptionally activated genes involved in hormone signaling (except for ethylene and salicylic acid signaling) were downregulated in abscised fruitlets. The differential expression of transcripts coding for enzymes involved in sucrose, glucose, trehalose, starch, galactose, and galactinol metabolism shows that sucrose, galactinol, and glucose synthesis and starch content were reduced as starch biosynthesis was blocked, and retrogradation and degradation intensified. These results suggest that the abscised pecan fruitlets stopped growing and developing for some time before dropping, further indicating that their sugar supply was reduced or stopped. The transcriptome characterization described in this paper contributes to unravelling the molecular mechanisms and pathways involved in the physiological abscission of pecan fruits.

Fungicide Resistance in Venturia effusa, Cause of Pecan Scab: Current Status and Practical Implications.

Pecan scab, caused by Venturia effusa, is the most economically damaging disease of pecan in the southeastern United States, and annual epidemics are most effectively managed through multiple fungicide applications. The fungicide applications can be the single greatest operating cost for commercial growers and the return on that investment is impacted by fungicide resistance. V. effusa produces multiple generations of conidia per season, exhibits substantial genetic diversity, overwinters as stromata in the tree, and is under immense selection from the applied fungicides, all of which lead to a high risk for developing fungicide resistance. Since the mid-1970s, resistance or reduced sensitivity has been observed in isolates of V. effusa to the methyl benzimidazole carbamates, demethylation inhibitors, quinone outside inhibitors, organotin compounds, and the guanidines. Over the last 10 years, several studies have been conducted that have improved both scab management and fungicide resistance management in V. effusa. The aim of this review is to summarize recent developments in our understanding of fungicide resistance in V. effusa in the context of scab management in southeastern pecan orchards. The history, modes of action, general use of the labeled fungicides, and mechanisms and stability of fungicide resistance in V. effusa are discussed; conclusions and future research priorities are also presented.

Identification and Analysis of the AP2 Subfamily Transcription Factors in the Pecan (Carya illinoinensis).

The AP2 transcriptional factors (TFs) belong to the APETALA2/ ethylene-responsive factor (AP2/ERF) superfamily and regulate various biological processes of plant growth and development, as well as response to biotic and abiotic stresses. However, genome-wide research on the AP2 subfamily TFs in the pecan (Carya illinoinensis) is rarely reported. In this paper, we identify 30 AP2 subfamily genes from pecans through a genome-wide search, and they were unevenly distributed on the pecan chromosomes. Then, a phylogenetic tree, gene structure and conserved motifs were further analyzed. The 30 AP2 genes were divided into euAP2, euANT and basalANT three clades. Moreover, the cis-acting elements analysis showed many light responsive elements, plant hormone-responsive elements and abiotic stress responsive elements are found in CiAP2 promoters. Furthermore, a qPCR analysis showed that genes clustered together usually shared similar expression patterns in euAP2 and basalANT clades, while the expression pattern in the euANT clade varied greatly. In developing pecan fruits, CiAP2-5, CiANT1 and CiANT2 shared similar expression patterns, and their expression levels decreased with fruit development. CiANT5 displayed the highest expression levels in developing fruits. The subcellular localization and transcriptional activation activity assay demonstrated that CiANT5 is located in the nucleus and functions as a transcription factor with transcriptional activation activity. These results help to comprehensively understand the pecan AP2 subfamily TFs and lay the foundation for further functional research on pecan AP2 family genes.

Photosystem Disorder Could be the Key Cause for the Formation of Albino Leaf Phenotype in Pecan.

Pecan is one of the most famous nut species in the world. The phenotype of mutants with albino leaves was found in the process of seeding pecan, providing ideal material for the study of the molecular mechanisms leading to the chlorina phenotype in plants. Both chlorophyll a and chlorophyll b contents in albino leaves (ALs) were significantly lower than those in green leaves (GLs). A total of 5171 differentially expression genes (DEGs) were identified in the comparison of ALs vs. GLs using high-throughput transcriptome sequencing; 2216 DEGs (42.85%) were upregulated and 2955 DEGs (57.15%) were downregulated. The expressions of genes related to chlorophyll biosynthesis (HEMA1, encoding glutamyl-tRNA reductase; ChlH, encoding Mg-protoporphyrin IX chelatase (Mg-chelatase) H subunit; CRD, encoding Mg-protoporphyrin IX monomethylester cyclase; POR, encoding protochlorophyllide reductase) in ALs were significantly lower than those in GLs. However, the expressions of genes related to chlorophyll degradation (PAO, encoding pheophorbide a oxygenase) in ALs were significantly higher than those in GLs, indicating that disturbance of chlorophyll a biosynthesis and intensification of chlorophyll degradation lead to the absence of chlorophyll in ALs of pecan. A total of 72 DEGs associated with photosynthesis pathway were identified in ALs compared to GLs, including photosystem I (15), photosystem II (19), cytochrome b6-f complex (3), photosynthetic electron transport (6), F-type ATPase (7), and photosynthesis-antenna proteins (22). Moreover, almost all the genes (68) mapped in the photosynthesis pathway showed decreased expression in ALs compared to GLs, declaring that the photosynthetic system embedded within the thylakoid membrane of chloroplast was disturbed in ALs of pecan. This study provides a theoretical basis for elucidating the molecular mechanism underlying the phenotype of chlorina seedlings of pecan.