Walnut Fruit Research Articles

Allometry and volumes in a nutshell: Analyzing walnut morphology using three‐dimensional X‐ray computed tomography

AbstractPersian walnuts (Juglans regia L.) are the second most produced and consumed tree nut, with over 2.6 million metric tons produced in the 2022–2023 harvest cycle alone. The United States is the second largest producer, accounting for 25% of the total global supply. Nonetheless, producers face an ever‐growing demand in a more uncertain climate landscape, which requires effective and efficient walnut selection and breeding of new cultivars with increased kernel content and easy‐to‐open shells. Past and current efforts select for these traits using hand‐held calipers and eye‐based evaluations. Yet there is plenty of morphology that meets the eye but goes unmeasured, such as the volume of inner air or the convexity of the kernel. Here, we study the shape of walnut fruits based on X‐ray computed tomography three‐dimensional reconstructions. We compute 49 different morphological phenotypes for 1264 individual nuts comprising 149 accessions. These phenotypes are complemented by traits of breeding interest such as ease of kernel removal and kernel‐to‐nut weight ratio. Through allometric relationships, relative growth of one tissue to another, we identify possible biophysical constraints at play during development. We explore multiple correlations between all morphological and commercial traits and identify which morphological traits can explain the most variability of commercial traits. We show that using only volume‐ and thickness‐based traits, especially inner air content, we can successfully encode several of the commercial traits.

Controlled atmosphere storage alleviates the browning of walnut (Juglans regia L.) fruit through enhancing GABA-mediated energy metabolism

Browning is the primary physiological damage that shortens the postharvest life and deteriorates the quality of stored fruits. Controlled atmosphere (CA) storage has been shown to be effective in alleviating the browning of ‘Xifu 1′ in-hull walnut; however, its alleviation mechanism is less understood. To further explore the effect and regulatory mechanism of CA on the browning of fresh in-hull walnut, we stored four varieties of in-hull walnuts under CA-treated (5% O2 + 7.5% CO2) and untreated conditions at 0 ± 0.5 °C with 70–80% relative humidity for 70 d. The indicators related to browning and γ-aminobutyric acid (GABA) metabolism were measured until 56 d of storage. The results showed that the CA significantly increased GABA levels by maintaining higher activities of glutamate decarboxylase (GAD), GABA transaminase (GABAT) and succinate dehydrogenase (SDH). This reduced the changes in the browning index (BI), color values, and respiration rate (RR) of the four walnut fruit varieties compared with controls during storage. Consistent with the active GABA metabolism, higher energy charge (EC) and adenosine triphosphate (ATP), succinic acid (SA), malic acid (MLA), fumaric acid (FA), and citric acid (CTA) contents were also observed in the CA-treated walnut hull compared to the controls. The epicarp degradation of the CA-treated ‘Xifu 1′ walnut fruit occurred later, and their mitochondria and cells maintained better integrity compared to the controls. Among the differentially expressed genes (DEGs) screened via RNA-Sequencing (RNA-Seq), 32 were annotated as part of the citrate cycle (TCA cycle) pathway, and 27 were annotated in the GABA metabolism pathway. Real-time quantitative PCR (RT-qPCR) analysis showed that eight candidate genes were closely related to the effect of CA on the synthesis, transport, and transformation of GABA. In conclusion, CA effectively controlled the browning of different varieties of fresh in-hull walnuts by enhancing GABA metabolism and maintaining energy balance.

Colletotrichum species (Glomerellales, Glomerellaceae) causing walnut anthracnose in China.

Colletotrichum species can function as plant pathogens, saprobes or endophytes on a wide variety of plant hosts and are considered amongst the ten most significant genera of plant pathogens globally. China contributes almost half the walnut production in the world. However, Colletotrichum species occurring on walnut remain largely unresolved in China. To explore the Colletotrichum species found on walnut in China, 470 walnut fruit or leaf samples with anthracnose were collected from 14 main walnut-producing regions across seven provinces. A total of 165 Colletotrichum strains were isolated from these samples. The Colletotrichum isolates were identified, based on morphological characteristics and sequence analyses of ACT, CHS-1, GAPDH, ITS and TUB2. Twelve species, including 11 known Colletotrichum species (C.boninense, C.citrulli, C.fioriniae, C.fructicola, C.godetiae, C.juglandicola, C.karsti, C.mengyinense, C.pandanicola, C.peakense and C.siamense) and a novel species (C.chinensis sp. nov.) were identified. The species distribution revealed regional prevalence as follows: C.mengyinense was the most dominant species in Gansu, C.mengyinense and C.siamense in Shandong, C.chinensis in Beijing, C.pandanicola in Shaanxi and C.godetiae in Yunnan. Colletotrichumsiamense was the sole species isolated in Sichuan and Xinjiang Provinces. Koch's postulates were fulfilled, demonstrating that all 12 species cause anthracnose on walnut. This is the first report of C.boninense, C.citrulli and C.karsti as pathogens of walnut anthracnose worldwide.

Assessment of the in-vitro toxicity and in-vivo therapeutic capabilities of Juglans regia on human prostate cancer and prostatic hyperplasia in rats

Benign prostatic hyperplasia (BPH) and prostatic cancer are aging-associated urological conditions in men. While significant advances have been made in relation to treatment, poor response, treatment resistance and adverse side-effects limit currently available therapies. The possible antiproliferative and/or ameliorative potentials of Juglans regia have been reported, however, there is a dearth of scientific information on its effect on BPH or prostatic cancer. In this study, in-vivo and in-vitro studies were used to assess the possible benefits of fresh walnut fruits extract in BPH or prostate cancer. High-performance liquid chromatography coupled with electro spray ionization and quadrupole-time-of-flight mass spectrometry (HPLC-ESI-Q-TOF) was used to assess the chemical profile of the plant extract. The anti-proliferative activity of Juglans regia extract was tested against normal and cancerous human prostate cell lines by using the iCELLigence real-time and label-free cell analysis system. The ameliorative potential of fresh fruits Juglans regia extract was assessed by administering the extract at 50 and 100 mg/kg body weight to rats with testosterone induced BPH. Analysis of phytochemicals in Juglans regia revealed a high concentration of phenolic acids and flavonoids, Juglans regia also showing time- and dose-dependent anti-proliferative activity against prostate cancer cells, and reversed biochemical and histomorphological changes induced by testosterone-induced BPH.

Identification of Botryosphaeria dothidea and Diaporthe eres from rotted walnut fruits and other plant parts in different phenological stages

AbstractThe acreage of English walnut (Juglans regia L.) is constantly expanding in Hungary, due to the favorable climatic conditions and economic importance. Last years, serious damage was reported from several orchards with high percentage of rotted, moldy kernels. The aim of this research was to identify the pathogens at different growth stages. Fungi were cultured from the spotty, shriveled and rotted kernels, and monosporic isolates were identified based on morphological characters and molecular markers (ITS region and tef1 locus sequences). Botryosphaeria dothidea and Diaporthe eres were identified in high proportion from symptomatic kernels. These species were also isolated from different parts of walnut trees in different seasons. D. eres was detected in a high proportion from asymptomatic buds in March, while the presence of both species was observed in symptomatic husks with Overnight Freezing-Incubation Technique (ONFIT) in June. Their optimal growth temperature defined to be between 20–25 °C, and the growth of D. eres isolates was completely inhibited at 35 °C.

The Determination of Mycotoxins in the Composition of Walnut (Juglans Regia L.) Fruits Cultivated in Different Geographical Regions of Uzbekistan by High–Performance Liquid Chromatography Method and Their Comparative Analysis

Mycotoxins are secondary metabolites produced by various fungi and are known to have significant adverse effects on human and animal health. If food products are contaminated with mycotoxins, their toxicity can cause various diseases. In this scientific research work, residual mycotoxins in nutrients were combined with liquid–liquid extraction (LLE) and analyzed through the use of reversed–phase high–performance liquid chromatography (RP–HPLC). These scientific studies were conducted in the experimental biology laboratory of Gulistan State University to determine the amount of aflatoxin (AFB2, AFG1, AFG2) in the samples of walnuts cultivated in six regions of Uzbekistan. The study revealed the detection of aflatoxins AFG1 type 0.003 in the designated region labeled as S20, AFB2 and AFG2 0.001 in J25, and AFG2 0.002 in S30. However, it is important to note that these levels did not above the maximum residue level standards established by the Republic of Uzbekistan. However, the aflatoxins produced by fungi are released based on the development of fungi at a temperature of 25–3000C, therefore it is recommended to store walnut fruits in dry conditions at a temperature of 1500C. All the methods used during scientific analysis can demonstrate high sensitivity and accuracy. All methods are successfully used to identify mycotoxins in the composition of walnut fruits, separate them from fungi and specify the amount of ingredients.

Walnut fruit diaphragm ethanol extract ameliorates damage due to Triton WR‐1339‐induced hyperlipidemia in rats

AbstractThe walnut fruit diaphragm (WFD) is an economically and ecologically important by‐product of the Juglandaceae family. Components in the ethanolic extract of WFD (E‐WFD) were identified by gas chromatography‐mass spectroscopy (GC‐MS). In our study, the lipid‐lowering effect, antioxidant potential, and anti‐inflammatory properties of E‐WFD in the blood, heart, and liver tissues of rats (♂, Wistar) with hyperlipidemia caused by Triton WR‐1339 were determined using biochemical, histopathological, and molecular techniques. Experimental animals for analysis are as follows: K1: healthy control group, K2: E‐WFD (150 mg, o.d.), K3: E‐WFD (300 mg, o.d.), K4: HL (hyperlipidemic group) (400 mg kg–1 Triton WR‐1339, i.p.), K5: HL + E‐WFD (300 mg) grouped as (n = 6). After the application, anti‐hyperlipidemic analysis (TC, TG, LDL‐C, HDL‐C, ALT, and AST), oxidative stress (MDA, SOD, and CAT) parameters measurement, histopathological examinations, and inflammatory biomarker genes (Tnf‐α, Il‐6, Il‐1β, Mmp‐2, Mmp‐9, Lxr‐α and Srebp‐1c) expression stability were determined by q‐RT PCR. With the E‐WFD application, there was a decrease in TC and TG levels and an increase in HDL‐C. Oxidative stress decreased by changing MDA, SOD, and CAT levels. Histopathological deterioration in the tissues changed positively. In addition, E‐WFD showed significant inhibitory properties on Tnf‐α, Il‐6, Il‐1β, Mmp‐2, Mmp‐9, Lxr‐α, and Srebp‐1c genes.Practical Applications: It is known that hyperlipidemia causes a wide range of diseases. In this study, the effect of walnut fruit diaphragm (WFD) ethanol extract was compared against the effect of Triton WR‐1339‐induced hyperlipidemia on rats. With the use of WFD, the damages caused by hyperlipidemia were significantly ameliorated. Accordingly, it is expected to contribute to the pharmacology, food and cosmetic industries as a very valuable alternative product.]

First report of walnut anthracnose caused by Colletotrichum orientalis in South Korea.

Walnut (Juglans regia L.) is known as "hodu" in South Korea. It is cultivated as a cash crop and an ornamental plant across South Korea. Walnut anthracnose is one of the main limiting factors for walnut production. In June 2022, severe anthracnose was observed in several walnut fields in Gimcheon, South Korea. The disease incidence was ~35% and symptoms on affected fruits consist of necrotic and circular lesions with or without conidial mass. Six pure isolates with similar morphological characteristics were obtained from the tissue of six affected fruits. Diseased tissues were cut into 3-to-4 mm2 pieces, surface sterilized by 1% sodium hypochlorite (NaOCl) for 2 minutes, and washed three times in sterile distilled water, then dried by blotting. Four pieces/plates were placed onto potato dextrose agar (PDA; Difco Becton Dickinson) and incubated at 25°C in the dark for 2-3 days. To obtain pure isolates, hyphal tips were transferred onto fresh PDA. The colonies of all isolates on the PDA were light gray to pinkish on the upper side and pink to vinaceous on the reverse after 7 days of incubation at 25°C in the dark. The conidia were hyaline, fusiform, aseptate, and 11.3 to 20.1 µm × 3.6 to 6.9 µm (n =100). Appressoria were single, cylendrical, smooth-walled, dark brown, and 8.1 to 14.3 × 5.4 to 10.5 μm (n =30). The morphological characteristics of the isolates were comparable with those of Colletotrichum orientalis (Chen et al. 2022). Three isolates (WN23, WN28 and WN30P) were selected based on morphological characteristics for molecular identification. The beta-tubulin (TUB2), actin (ACT), chitin synthase 1 (CHS-1), and histone 3 (HIS3) genes of selected isolates were amplified and sequenced using the primers Bt2a/Bt2b, ACT512F/ACT783R, CHS-79F/CHS-345R and CYLH3F + CYLH3R, respectively (Damm et al. 2012). The resulting consensus sequences of each gene were deposited in GenBank with the accession numbers (TUB2: LC742910 to LC742912; CHS-1: LC742916 to LC742918; ACT: LC742919 to LC742921, HIS3: LC742922 to LC742924). The constructed maximum likelihood tree based on TUB2, ACT, CHS-1, and HIS3 sequence data revealed that the three isolates formed a clade with C. orientalis reference strain CBS 128532 (ex-type). The pathogenicity of two representative isolates (WN28 and WN30P) was confirmed on healthy, surface-sterilized, and detached walnut fruits. Both wounded and nonwounded fruits (10/isolate) were inoculated with the conidial suspension (106 spores/ml). The wounded and unwounded control fruits were inoculated with sterile distilled water. All the fruits (inoculated and control) were incubated in containers at 25°C with a 12-h/12-h light/dark cycle. This experiment was repeated twice. Necrotic lesions were observed in all inoculated wounded fruits, while unwounded and control fruits remained asymptomatic. The pathogen was re-isolated from inoculated fruits and identified as C. orientalis by morphological characteristics and ITS and TUB2 sequences. Damm et al. (2012) has identified C. fioriniae as species belongs to two well separated clades. However, Chen et al. (2022) split the C. fioriniae species into two species, with the new species called C. orientalis, as a causal agent of apple bitter rot. In previous studies, C. fioriniae has been reported as a causal of walnut anthracnose (Luongo et al. 2021; Zhu et al. 2015; Varjas et al. 2019). To our knowledge, this is the first report of C. orientalis as the causal agent of walnut anthracnose in South Korea. Since this disease reduces the commercial value and quality of walnut fruits, effective management practices should be developed to deal with C. orientalis.

The Effects of Altitude on Fruit Characteristics, Nutrient Chemicals, and Biochemical Properties of Walnut Fruits (Juglans regia L.)

This study aimed to investigate changes in fruit characteristics, total phenolics, total antioxidant capacity, organic acids, sugar content, and fatty acid composition in ten walnut genotypes and three cultivars grown at different altitudes under Mediterranean climate conditions. At altitudes of 500 m and 1200 m, total phenolics ranged between 237.51 and 412.96 mg GAE/100 g dw and 234.94 and 392.31 mg GAE/100 g dw, respectively. According to DPPH radical inhibition, the total antioxidant capacity varied between 47.65% and 64.99% at an altitude of 500 m and between 57.76% and 68.92% at an altitude of 1200 m. The oil content demonstrated variations between 53.44% and 76.17% at an elevation of 500 m and between 43.11% and 67.03% at an altitude of 1200 m. At both 500 m and 1200 m altitudes, linoleic acid emerged as the predominant fatty acid and ranged from 58.82% to 62.44% at 500 m altitude and from 57.33% to 59.38% at 1200 m altitude. Notably, malic acid was identified as the primary organic acid, with concentrations ranging from 1.35% to 7.33% at both altitudes. In conclusion, this study demonstrates that walnut seeds are abundant in oil, protein, total phenolics, antioxidants, and fatty acids, with variations influenced by the cultivar or genotype and notably affected by altitude.

Comprehensive agrobiological assessment and analysis of genetic relationships of promising walnut varieties of the Nikitsky Botanical Gardens.

Walnut is an important horticultural crop, the production of which ranks second among all nut crops. Despite the significant demand in the domestic market in Russia, the industrial production of walnut fruits in Russia is currently underdeveloped. At the same time, there is a need to update the assortment with new highly productive varieties adapted to local agro-climatic conditions and having high quality nuts that are competitive at the world level. An important issue for the successful implementation of breeding programs is a comprehensive study of the gene pool. In this regard, within the framework of the study, the task was to evaluate promising varieties from the collection of the walnut gene pool of the Nikitsky Botanical Gardens and analyze genetic relationships based on microsatellite genotyping. On the basis of the performed phenotypic assessment, the study sample, which included 31 varieties, was divided into several groups according to the main phenotypic traits, such as frost and drought resistance, the start of the growing season, the ripening period, the weight and type of flowering, the weight of the fruit, and the thickness of the endocarp. Varieties with economically valuable traits that can be recommended as promising as initial parental forms in breeding work for resistance to abiotic stress factors have been identified, as well as varieties with increased productivity and large fruit sizes. Based on the analysis of eight SSR markers (WGA001, WGA376, WGA069, WGA276, WGA009, WGA202, WGA089 and WGA054), an analysis of the level of genetic diversity was performed and genetic relationships were established in the studied sample of varieties. Six (for WGA089) to eleven (for WGA276) alleles per locus have been identified. A total of 70 alleles were identified for the eight DNA markers used, with an average value of 8.75. Analysis of SSR genotyping data using Bayesian analysis established the presence of two main groups of genotypes. Taking into account the fact that all the studied varieties are selections from local seed populations in different regions of the Crimean Peninsula, the revealed level of polymorphism may indirectly reflect the level of genetic diversity of the local walnut populations. Furthermore, the presence of two genetically distant groups indicates the presence of two independently formed pools of the autochthonous gene pool of the species Juglans regia L. on the Crimean Peninsula.

Effects of cultivar susceptibility, fruit maturity, and natural wounds on the infection of English walnut (Juglans regia L.) fruits by Botryosphaeriaceae and Diaporthe fungi

Effects of cultivar susceptibility, fruit maturity, and natural wounds on the infection of English walnut (Juglans regia L.) fruits by Botryosphaeriaceae and Diaporthe fungi

First Report of Colletotrichum godetiae Causing Anthracnose on Walnut (Juglans regia and Juglans sigillata) in China.

In August 2020, anthracnose lesions were observed on fruits of Juglans regia and J. sigillata in walnut orchards, in Yijun (Shaanxi Province) and Nanhua (Yunnan Province) counties, China. Symptoms on walnut fruits first appeared as small necrotic spots that rapidly enlarged into subcircular or irregular sunken black lesions (Fig. 1a, b). Sixty diseased walnut fruits (30 fruits of J. regia and J. sigillata, respectively) were randomly sampled from six orchards (10-15 ha each orchard, three orchards were selected in each county) with severe anthracnose (incidence rate of fruit anthracnose is over 60% in the orchard.) in two counties. Twenty-six single spore isolates were obtained from diseased fruits as described by Cai et al. (2009). After seven days, isolates formed grey to milky white colony with abundant aerial hyphae on the upper surface of colony, and milky white to light olive on the back of PDA (Fig. 1c). Conidiogenous cells were hyaline, smooth-walled, and cylindrical to clavate (Fig. 1d). Conidia were smooth-walled, aseptate, cylindrical to fusiform, with both ends acute or one end round and one end slightly acute (Fig. 1e), and ranged in size from 15.5-24.3×4.9-8.1 µm (n=30). Appressoria were brown to medium brown, clavate to elliptical, with the edge entire or undulate (Fig. 1f), and ranged in size from 8.0-27.6×4.7-13.7μm (n=30). The morphological characteristics of 26 isolates were similar to those of the species complex Colletotrichum acutatum (Damm et al. 2012). Six representative isolates were randomly selected (three isolates for each province) for molecular analysis. The ribosomal internal transcribed spacers (ITS) (White et al. 1990), beta-tubulin (TUB2) (Glass and Donaldson 1995), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Templeton et al.1992) and chitin synthase 1 (CHS-1) (Carbone and Kohn 1999) genes were amplified and sequenced. Sequences of 6 of 26 isolates were submitted to GenBank (Accession Nos: ITS: MT799938-MT799943, TUB: MT816321-MT816326, GAPDH:MT816327-MT816332, CHS-1: MT816333-816338). Multi-locus phylogenetic analyses revealed that six isolates clustered together with Colletotrichum godetiae ex-type culture isolates CBS133.44 and CBS130251, and the bootstrap support value was 100% (Fig.2). The pathogenicity of two representative isolates (CFCC54247 and CFCC54244) was tested using healthy fruits of the " J. regia cv. Xiangling" and " J. sigillata cv. Yangbi" varieties. Forty sterilized fruits (20 fruits were inoculated with CFCC54247, and 20 fruits with CFCC54244) were wounded by puncturing with a sterile needle through walnut pericarp and inoculated in the wound site with 10 μl of conidial suspension (106 conidia/ml) from seven day old colonies grown on PDA at 25℃. Twenty wounded fruits were inoculated with sterile water as control. Inoculated and control fruits were incubated in containers at 25℃ in a 12/12h light/dark cycle. The experiment was repeated three times. Anthracnose symptoms (Fig. 1g-h) were observed in all inoculated fruits after 12 days, whereas controls showed no symptoms. Fungal isolates from inoculated diseased fruits showed the same morphological and molecular characteristics as the isolates obtained in this study, confirming Koch's postulates. To our knowledge, this is the first report of C. godetiae causing anthracnose on the two walnut species in China. The result will be helpful for providing a basis for further research on the control of the disease.

Association of Rahnella victoriana, Enterobacter hormaechei subsp. hoffmannii and Citrobacter braakii with walnut decline

Persian walnut (Juglans regia) has a considerable economic importance worldwide. However, the vigor and vitality of walnut trees were heavily affected by bark canker during the last few years. Irregular longitudinal cankers in the outer bark, stem tissue necrosis, and bleeding with black-colored exudates walnut trees were observed in Kermanshah, Hamedan, Markazi, Alborz, Isfahan, Qom, Semnan, and Razavi Khorasan provinces in western, central and eastern Iran during 2018 and 2019. A total of 150 symptomatic samples were collected from affected walnut trees in order to identify bacteria associated with walnut decline. Two-hundred sixty strains with a metallic green sheen were isolated on EMB-agar medium. The pathogenicity of all strains was proved by inoculating a suspension of the bacterial strains under the bark of immature walnut fruits cv. ‘Hartley’. Ninety-five strains caused necrosis and a dark-colored region in the mesocarp around the inoculation site 14 days post-inoculation. Moreover, 12 representative strains induced necrotic and black-colored tissues in the bark of young green twigs of two-year old walnut seedling cv. ‘Chandler’. The strains were classified into four categories based on conventional phenotypic characters confirmed with the 16S rRNA gene sequences. A phylogenetic tree based on the concatenated sequences of two housekeeping gene fragments, gyrB and infB, indicated that strains including I1, Q6, and S6 were grouped in a cluster with Gibbsiella quercinecans FBR97T as well as strains I2, I5, and KE6 were clustered with Rahnella victoriana FRB 225T. Moreover, strains MR1, MR3, and MR5 were grouped with the Enterobacter hormaechei subsp. hoffmannii DSM 14563T. The phylogenetic analyses based on the partial sequencing of housekeeping genes including fusA, pyrG, and leuS revealed that strains KH1, KH3, and KH7 belong to Citrobacter braakii species. To the best of our knowledge, this is the first report of C. braakii and E. hormaechei as plant pathogens and R. victoriana associated with walnut decline.

Role of the Green Husks of Persian Walnut (Juglans regia L.)—A Review

Green husks are the outer layer of walnut fruits. They form part of the agro-residues discarded away upon nut maturity in the walnut industry. Although it is not used, research studies show that green husk is a rich source of natural bioactive phytochemicals. A total of 83 individual phenolic compounds were identified in walnut husks, mainly consisting of naphthoquinones, flavonols, and hydroxycinnamic acids. There is no standard profile of phenolic compounds in walnuts; the quantity and quality of phenolic compounds vary by cultivar. Walnut extracts exhibit strong antioxidant activities and play an important role in the plant’s defence mechanisms against diseases, attacking different plant organs. The study provides a review of total phenolic content and individual phenolic compounds identified in green husks of different walnut cultivars as documented in different studies. It also explores the role and application of green husks in various industries such as traditional medicine, textile, wood, leather, beverage, and agriculture. In addition, the effects of phenolic compounds on biotic and abiotic factors are also evaluated.

Metabolomics and Transcriptomics Analyses Reveals the Molecular Regulatory Mechanisms of Walnut (Juglans regia L.) Embryos in Response to Shade Treatment.

The walnut is an important nut that has numerous uses worldwide. However, due to dwarf and close plantation methods as well as continuous cloudy or rainy days that occur during periods of walnut oil accumulation, the walnut fruit exhibits varying degrees of stress under low-light conditions. However, the effects of shade on metabolites and genes in walnut embryos remain unclear in the literature. The purpose of this study is to investigate the lipid biosynthesis process that occurs in walnut embryos under shade treatment via the use of metabolomics and transcriptomics analyses. The results indicate that the oil content decreases significantly under shaded conditions, while the protein content increases significantly. The expression levels of fatty acid desaturase 2 (FAD2) and stearoyl-ACP-desaturase (SAD) involved in the lipid biosynthesis mechanism were significantly reduced in the shaded group, which resulted in reductions in oleic (C18:1), linoleic (C18:2), and α-linolenic (C18:3) acids. The reduced oil content was consistent with the downregulation of genes associated with the lipid biosynthesis mechanism. In the amino acid biosynthesis process, the upregulated cysteine synthase (cscK) and anthranilate synthase beta subunit 2 (trpG) genes promoted the accumulation of L-aspartic acid and L-citrulline. The increase in protein content was consistent with the upregulation of genes related to amino acid biosynthesis. Thus, our study provides new insights into the regulatory mechanisms of shade underlying overall walnut fruit quality.

Estimation of Nitrogen Concentration in Walnut Canopies in Southern Xinjiang Based on UAV Multispectral Images

Nitrogen is one of the most important nutrients affecting the growth and fruit quality of walnut trees. Rapid and accurate monitoring of nitrogen concentration in the walnut canopy can provide a digital basis for its nutritional diagnosis and precision fertilization. Consequently, the main purpose of this study was to use Unmanned Aerial Vehicle (UAV) remote sensing technology to monitor the nitrogen concentration of walnut canopies. In this study, UAV multispectral images of the canopies of nine walnut orchards with different management levels in Wensu County, South Xinjiang, China, were collected during the fast-growing (20 May), sclerotization (25 June), and near-maturity (27 August) periods of walnut fruit, and canopy nitrogen concentration data for 180 individual plants were collected during the same periods. The validity of the information extracted via the outline canopy and simulated canopy methods was compared. The accuracy of nitrogen concentration inversion for three modeling methods, partial least squares regression (PLSR), support vector machine (SVM), and random forest (RF), was analyzed; the effects of different combinations of variables on model accuracy were compared; and the spatial distribution of the nitrogen concentration in the walnut canopy was numerically mapped using the optimal model. The results showed that the accuracy of the model created using the single plant information extracted from the outlined canopy was better than that of the simulated canopy method, but the simulated canopy method was more efficient in extracting effective information from the single plant canopy than the outlined canopy. The simulated canopy method overcame the difficulty of mismatching the spectral information of individual plants extracted, by outlining the canopy in the original image for nitrogen distribution mapping with the spectral information of image elements in the original resolution image. The prediction accuracy of the RF model was better than that of the SVM and PLSR models; the prediction accuracy of the model using a combination of waveband texture information and vegetation index texture information was better than that of the single-source model. The coefficients of determination (R2) values of the RF prediction model built using the band texture information extracted via the simulated canopy method with the vegetation index texture information were in the range of 0.61–0.84, the root mean square error (RMSE) values were in the range of 0.27–0.43 g kg−1, and the relative analysis error (RPD) values were in the range of 1.58–2.20. This study shows that it is feasible to monitor the nitrogen concentration of walnut tree canopies using UAV multispectral remote sensing. This study provides a theoretical basis and methodological reference for the rapid monitoring of nutrients in fruit trees in southern Xinjiang.

Analysis of phenolic compounds in black walnut (Juglans nigra L.) fruit extract using high-performance liquid chromatography

Black walnut (Juglans nigra L.), which belongs to the Nut family (Juglandaceae), is a valuable raw material for the production of phytopreparations. The extract from the fruits of Juglans nigra L. has immunomodulating and antioxidant properties, which is primarily due to the presence of a large amount of phenolic compounds in the plant. But the phytochemical composition and pharmacological properties of Juglans nigra L. have not been sufficiently studied.
 The aim of our work was to study the optimal conditions for the extraction of Juglans nigra L. fruits, followed by the identification of its quantitative and qualitative composition.
 The main criterion for evaluating optimal extraction conditions was the quantitative content of phenolic compounds, which was determined spectrophotometrically by the Folin–Chocalteu method. The analysis of polyphenolic compounds was carried out by the method of high performance liquid chromatography.
 On the basis of experimental studies, the optimal conditions for extracting the fruits of Juglans nigra L. by maceration method conditions were selected. According to the results of the comparative analysis, it is shown that for the maximum extraction of phenolic compounds, it is advisable to use a 70% water-ethanol mixture as an extractant with a raw material to solvent ratio of 1:9. The method of high-performance liquid chromatography showed that the total content of phenolic compounds in the fruits of Juglans nigra L. is 2 327.09 μg/ml. Analysis of the extract of Juglans nigra L. by high-performance liquid chromatography method showed the presence of such phenolic compounds in plant material samples as catechins, catechin-like substances, flavonols (rutin, quercetin, myricetin), flavanones (naringin, naringenin) and phenolic acids. It should be noted, that the presence of such substances as chlorogenic acid, caffeic acid, kaempferol, hesperidin, genistein and daidzein glycosides, luteolin and apigenin glycosides, luteolin and apigenin in the extract of Juglans nigra L. was not detected.
 A high content of compounds of polyphenolic nature in the fruits of Juglans nigra L. was established. Therefore, this medicinal plant material is promising for further research, in particular, for the development of medicines based on the extract with potential anti-inflammatory, analgesic and antioxidant activity.

Re-sequencing and transcriptomic analysis reveal differential expression patterns and sequence variation in glucosyltransferase gene related to anthocyanin biosynthesis in walnut ( Juglans regia L.)

The color of walnut peel, usually green during the development stages, is one of the important appearance qualities of walnut fruit. Red walnut ‘RW-1’ with red peel is a specific germplasm resources due to anthocyanin accumulation, but the mechanism remains unclear. Here, re-sequencing and transcriptome were used to analyze the differences of anthocyanin accumulation in walnut peel with different colors. There were 2,696,620 SNPs and 495,238 InDels between ‘Zhonglin 1’ and ‘RW-1’, and the genetic relationship between them was closer than that of either and walnut reference genome ‘Chandler’. 1,156 differentially expressed genes (DEGs) with structural polymorphisms were screened by combining re-sequencing and transcriptome analysis. LOC108991235 (JrUFGT5), encoding anthocyanidin 3-O-glucosyltransferase, was identified by KEGG function enrichment. The JrUFGT5 gene base substitutions identified in the coding sequence (CDS) region resulted in amino acid variation, but did not occur in the protein domain region. The expression pattern of JrUFGT5 gene was positively correlated with the anthocyanin accumulation in different walnut peels, and the subcellular localization analysis showed JrUFGT5 was located in the cytoplasm and nucleus. Collectively, these findings revealed the differential expression patterns and sequence variation of a crucial anthocyanidin 3-O-glucosyltransferase gene in walnuts with different colors, which is of great significance to the exploration of the mechanism of anthocyanin biosynthesis in red walnut.

No Evidence of an Elevation Effect Caused by Temperature Differences on Soil Microbial Properties in a Walnut Fruit Forest in Kyrgyzstan

This study is to investigate the effect of differences in annual average temperature on soil microbial communities as caused by elevation in walnut-fruit forests in Kyrgyzstan with similar vegetation. Soil samples (n = 10 per site) were collected from top- and subsoil at three elevation levels (1000, 1300 and 1600 m above sea level) with an average temperature difference of 1.3 °C between sites and analysed for soil chemical and biological properties. All soil properties showed high variability within, but most revealed no differences between elevation levels. Microbial biomass, activity and community composition were largely similar at all sites with slightly higher fungal contribution based on internal transcribed spacer (ITS) sequence counts at high elevation, which, however was not reflected by ergosterol. Total soil organic carbon and nitrogen levels did not show elevation effects either. Mehlich-extractable elements revealed positive relationship with soil microbial properties, which was in particular pronounced for copper, manganese and zinc, highlighting the relevance of trace elements for soil microorganisms. The subsoil showed lower levels for all microbial properties even though they were on a comparably high level; it contained smaller sized bacteria and fungi, as revealed by MBC/dsDNA ratios, and fungal ITS counts/ergosterol ratios illustrating growth limitations for microorganisms in subsoils. Elevation with long-term average temperature differences did not yield pronounced differences in soil microbial properties, which were more potentially stronger affected by similar C input quantity and substrate quality from the similar vegetation. Consequently, climate change effects will more likely affect microbial properties indirectly via changes in vegetation.

Understanding the association of apical necrosis in flowers and fruits of walnut (Juglans regia L.) with causal microorganisms under field conditions

Understanding the association of apical necrosis in flowers and fruits of walnut (Juglans regia L.) with causal microorganisms under field conditions