Alternate bearing in 'Hass' avocado - Fruit load-induced changes in bud auxin homeostasis are associated with flowering repression.

In 'Hass' avocado (Persea americana), fruit presence reduces next season flowering. Recent fruit tree studies proposed that heavy fruit load (HFL) generates an auxin (IAA) signal in the buds, which represses flowering. However, the nature of this signal remains unknown. Here, we investigated the effect of avocado HFL on bud IAA accumulation and flowering transition. We found that IAA-aspartate and IAA-glutamate conjugate levels were significantly higher in buds from 'on' (fully loaded) than 'off' (low-loaded) trees, hinting that free IAA levels were higher in the former. Expression analysis showed that coinciding with flowering reduction, HFL induced the floral repressor PaTFL1, and suggested that accumulation of IAA in buds as imposed by HFL was associated with its conjugation to aspartate and glutamate and resulted both from de novo IAA synthesis, as well as from reduced IAA export. Accordingly, experiments involving radiolabelled 14C-IAA demonstrated that HFL reduced shoot basipetal IAA transport. Lastly, we confirmed the negative effects of IAA on flowering, showing that IAA and PAT blocker (TIBA) treatments delayed 'off' trees inflorescence development, reducing their inflorescence axis and inducing PaTFL1 transcript. Together, our data suggest that avocado HFL generates IAA signalling in buds that induces PaTFL1, which represses inflorescence development.

Isohydric stomatal behaviour alters fruit vascular flows and minimizes fruit size reductions in drought-stressed 'Hass' avocado (Persea americana Mill.).

BACKGROUND AND AIMS: Plant water status is important for fruit development, because many fleshy fruits contain large amounts of water. However, there is no information on vascular flows of Persea americana 'Hass' avocado. The aims of this research were to explore the impact of drought stress on the water relationships of the 'Hass' avocado plant and its fruit growth. METHODS: Well-watered and water-stressed 'Hass' avocado plants were compared. Over 4 weeks, water flows through the shoot and fruit pedicel were monitored using external sap flow gauges. Fruit diameter was monitored using linear transducers, and stomatal conductance (gs), photosynthesis (A) and leaf and stem water potentials (Ñ°leaf and Ñ°stem) were measured to assess the response of the plants to water supply. KEY RESULTS: In well-watered conditions, the average water inflow to the shoot was 72 g day-1. Fruit water inflow was 2.72 g day-1, but there was water loss of 0.37 g day-1 caused by the outflow (loss back into the tree) through the vascular tissues and 1.06 g day-1 from the fruit skin. Overall, fruit volume increased by 1.4 cm3 day-1. In contrast, water flow into fruit of water-stressed plants decreased to 1.88 g day-1, with the outflow increasing to 0.61 g day-1. As a result, increases in fruit volume were reduced to 0.4 cm3 day-1. The values of A, gs and sap flow to shoots were also reduced during drought conditions. Changes in the hourly time-courses of pedicel sap flow, fruit volume and stem water potential during drought suggest that the stomatal response prevented larger increases in outflow from the fruit. Following re-watering, a substantial recovery in growth rate was observed. CONCLUSIONS: In summary, a reduction in growth of avocado fruit was observed with induced water deficit, but the isohydric stomatal behaviour of the leaves helped to minimize negative changes in water balance. Also, there was substantial recovery after re-watering, hence the short-term water stress did not decrease avocado fruit size. Negative impacts might appear if the drought treatment were prolonged.

Removal of fluoroquinolone antibiotic and sulfonated dye by functionalized Persea americana seed powder: Appraisal on phase transfer kinetics, equilibrium, economics, and applications in rural settings.

The study focuses on reactive orange 16 (RO16), a sulfonated dye, and ciprofloxacin (CiP), a fluoroquinolone antibiotic treatment from aquatic surface by adsorption. The functionalized Persea americana seed powder (PASP) was developed by acid hydrolysis technique and investigated for RO16 and CiP removal in batch scale at different concentrations for CiP and RO16, pH (2-8), contact duration and temperature (303-318K). Utilizing a scanning electron microscope (SEM) with energy dispersive X-ray spectroscopy (EDAX), the generated native PASP were assessed for their morphological characteristics. Fourier transform infrared (FTIR) spectroscopy was applied to examine the performing characteristics of PASP. Experimental findings with four kinetic mathematical models allowed the estimation of the process involved in the biosorption. The most effective agreement was explained by the pseudo-second-order model and Sips isotherm (Cip = 34.603 mg/g and RO16 = 30.357 mg/g) at 303K temperature. For Cip Process economics of the biosorbent was done, and it was observed that it was less than the readily market-available activated carbon.

First Report of Phytophthora cinnamomi causing root rot of avocado trees in Greece.

In September 2023, thirty declining 30-year-old avocado (Persea americana) trees ('Hass' grafted on 'Zutano' seedlings) were detected in a 1.5-ha orchard in the island of Crete (Chania region). Crown symptoms encompassed wilting and leaf chlorosis, advancing to defoliation and extensive dieback. Tap and feeder roots decayed and brown discoloration of root tissues was evident on heavily infected trees. The disease was severe and widespread, resulting in a 5% mortality rate among 300 trees. The pathogen was isolated with a modified soil baiting technique (Ferguson and Jeffers, 1999). Surface disinfected avocado fruits were immersed in water containing soil samples. Following a period of 2 to 8 days, tissue fragments from the resulting necrotic lesions on the fruit surface were transferred on ΡΑRP medium and subsequently incubated at 20°C (Ferguson and Jeffers, 1999). Three isolates (AV2, AV12 and AV11a) were obtained by transferring single hyphal tips to new Petri dishes containing V8 juice agar. They were grown at 20˚C and used for identification after 10 days. Isolates formed coralloid colonies with abundant clustered spherical hyphal swellings and terminal or intercalary (ratio 1:5) thick-walled chlamydospores measuring 20 to 36 µm (avg 29±0.8 µm) with characteristic thick walls (avg 1.2±0.2 µm). Sporangia, produced in non-sterile soil-extract water, were ovoid to obpyriform, persistent, non-papillate, 32 to 81 µm (avg 56±4.8 µm) long and 20 to 42 µm (avg 31±3.2 µm) wide (n=100). Isolates were heterothallic as they did not produce oospores in single cultures. Based on the morphological traits the isolates were identified as Phytophthora cinnamomi (Erwin and Ribeiro 1996). The internal transcribe spacer region (ITS) including ITS1, 5.8S rDNA region, and ITS2 as well as the cytochrome c oxidase subunit I (coxI) gene of the three representative isolates wereamplified with ITS1/ITS4 and FM83/FM84 primers, respectively (White et al. 1990; Martin and Tooley 2003), and sequenced (GenBank acc. PP506613 to PP506615 and PQ063867 to PQ063869, respectively). BLAST search revealed almost 100% identity with the sequences of P. cinnamomi ex-isotype isolate (KC478663 and KU899315 respectively). Pathogenicity tests using isolate AV2 were conducted following the soil infestation method (Jung et al. 1996) using six-year-old avocado 'Zutano' seedlings. Six non-inoculated plants treated with vermiculite-multivitamin juice mixture were used as controls. Plants (1 m tall) were grown in pots under greenhouse conditions and watered regularly. Six weeks post inoculation, all inoculated trees showed chlorosis, wilting and root rot, while control plants remained symptomless. Symptoms were similar to those observed in the field and the pathogen was re-isolated and molecularly identified as previously described. This study presents the first documented occurrence of P. cinnamomi, widely regarded as the most destructive avocado pathogen globally, on avocado crops in Greece (Rodger et al. 2019). Additionally, this marks the first recorded presence of this pathogen on the island of Crete, regardless of the host species. The accurate identification of Phytophthora species associated with avocado root rot is essential for implementing an effective disease management strategy, particularly in the selection of appropriate disease-resistant rootstocks.

Searching for laurel wilt resistance in avocados of Mexican and Mexican-Guatemalan ancestry.

Laurel wilt (LW), a lethal vascular disease caused by the ambrosia fungus Harringtonia lauricola, has severely reduced avocado (Persea americana Mill.) production in Florida and decimated populations of native lauraceous trees across twelve U.S. states. All commercial avocado cultivars evaluated to date succumb to the disease, but the speed at which the tree declines varies. Cultivars with West Indian (WI) genetic background develop severe symptoms faster than those with Mexican (M) and Guatemalan (G) pedigree. Genetic resistance to LW is urgently needed, as management relies on costly cultural practices. We screened non-commercial open-pollinated progenies from 19 Mexican and 6 Mexican x Guatemalan accessions, and 2 Guatemalan x West Indian cultivars recognized as tolerant by growers. From the five disease response parameters evaluated, the final disease intensity index and disease severity on the last evaluation day were used to classify genotypes. A wide variability of responses was observed within and among families. Symptomatic plants were present in all families, while some individuals within 15 families remained asymptomatic. 'Colín V-33' (M×G) family was identified as tolerant, and Libres 3 (M), Bladimiro M-06 (M) 'Colinmex' (M×G), 'Collinson' (G×WI), Libres 5 (M) and Rag-13 (M) families were classified as moderately tolerant. This is the first-time tolerance to laurel wilt in avocado is formally reported, though surviving material needs to be propagated for response validation and field testing. Identifying tolerant accessions can help understand the underlying mechanisms and provide breeders with genetic resources for the future incorporation of resistance genes into commercial cultivars.

First Report of Leaf Spot Disease on Avocado Caused by Bipolaris setariae in China.

Avocado (Persea americana), which is native to Latin America, is mostly planted in southwest China. In November 2021, leaf spot symptoms were observed in a nursery in Chongzuo (22.2019°N, 106.4723°E), Guangxi, China. Approximately 90% of avocado seedlings in the nursery were affected. Symptomatic plant fully expanded leaves showed small brown spots that ranged from 1 to 3 mm, with a yellow halo around (Fig.1). Lesions gradually expanded and became nearly round and dark brown. Finally, leaves withered or curled. For pathogen isolation, 15 symptomatic leaves were randomly sampled from different plants of the nursery, five leaves were selected and four samples size 4×4mm were taken from each leaf and were plated on potato glucose agar. Identical fungus colonies were observed in 80% of the samples, and no bacteria were isolated. Single conidial isolation was performed. After 4 days, the colony diameter reached 74.6 mm, colonies appeared gray, and developed aerial hyphae. Conidiophores were mostly solitary with a few clustered erect or slightly curved, knee shaped, and 3.89 to 5.24 µm wide. Conidia were 39.33 -96.88 × 9.96 - 15.59 µm, slightly curved, rarely straight, light brown to yellowish brown, fusoid or navicular, and truncated at the base with 4 to 10 septa. Based on morphological and cultural characteristics, the fungus was identified as Bipolaris sp. (Manamgoda et al. 2014). An isolate named MP211122 was grown on Sachs' ager at 27℃ under 12-h light/dark for 1 week and consistently with Adhikari et al. (2021) no sexual from was observed. To confirm the tentative identification, genomic DNA was extracted, ITS and GAPDH gene were amplified and sequenced using primers ITS1/ITS4 and GPD/GPD2, respectively (Tan et al. 2022). The ITS sequence (GenBank ON248469) shared 100% identity with B. setariae (MN215632.1), and the GAPDH sequence (ON642344) shared 99.82% identity with B. setariae (MF490833.1, MK144540.1) and B. yamadae (MK026428.1). A maximum likelihood phylogenetic analysis based on GAPDH and ITS sequences using MEGA 7.0 revealed that the isolate clustered with B. setariae with 100% bootstrap support(Fig. 2). Healthy 11-month old potted avocado seedlings from disease-free nursery were selected , the conidial suspension (1 × 105 conidia/mL) of MP211122 isolate was prepared by harvesting conidia from a 10-day-old culture on water agar. Conidia were sprayed onto young leaves of six potted plants. Three additional seedlings sprayed with sterile distilled water served as controls. All plants were covered with plastic bags for 3 days to maintain high humidity and then maintained in a greenhouse at 30℃ with a 12-h/12-h light/dark cycle. After 5 days, typical symptoms of small brown spots were observed on all inoculated leaves (Fig.3). All leaves on control plants remained asymptomatic. The reisolated fungus was morphologically identical to the original isolate used for inoculation, fulfilling Koch's postulates. This is the first report of B. setariae as a pathogen causing leaf spot on avocado in China. This information will facilitate further studies, monitoring and control of the disease as accurate identification of the causal agent is a primary requisite for designing management strategies.

Changes in the expression of genes encoding xanthophyl acyltransferases during the postharvest ripening of avocado (Persea americana) fruit.

BACKGROUND: Avocado fruit is rich in xanthophylls, which have been related to positive effects on human health. Xanthophyl acetyltransferases (XATs) are enzymes catalyzing the esterification of carboxylic acids to the hydroxyl group of the xanthophyll molecule. This esterification is thought to increase the lipophilic nature of the xanthophyll and its stability in a lipophilic environment. Studies on XATs in fruits are very scarce, and no studies had been carried out in avocado fruit during postharvest. The objective of this work was to investigate the changes in the expression of genes encoding XAT, during avocado fruit ripening. RESULTS: Avocado fruits were obtained from a local market and stored at 15 °C for 8 days. The fruit respiration rate, ethylene production, and fruit peel's color space parameters (L*, a*, b*) were measured during storage. Fruit mesocarp samples were taken after 1, 3, 5, and 7 days of storage and frozen with liquid nitrogen. Total RNA was extracted from fruit mesocarp, and the quantification of the two genes designated as COGE_ID: 936743791 and COGE_ID: 936800185 encoding XATs was performed with real-time quantitative reverse transcription polymerase chain reaction using actin as a reference gene. The presence of a climacteric peak and large changes in color were recorded during postharvest. The two genes studied showed a large expression after 3 days of fruit storage. CONCLUSIONS: We conclude that during the last stages of ripening in avocado fruit there was an active esterification of xanthophylls with carboxylic acids, which suggests the presence of esterified xanthophylls in the fruit mesocarp. © 2024 Society of Chemical Industry.

The expression of the NPR1-dependent defense response pathway genes in Persea americana (Mill.) following infection with Phytophthora cinnamomi.

A plant's defense against pathogens involves an extensive set of phytohormone regulated defense signaling pathways. The salicylic acid (SA)-signaling pathway is one of the most well-studied in plant defense. The bulk of SA-related defense gene expression and the subsequent establishment of systemic acquired resistance (SAR) is dependent on the nonexpressor of pathogenesis-related genes 1 (NPR1). Therefore, understanding the NPR1 pathway and all its associations has the potential to provide valuable insights into defense against pathogens. The causal agent of Phytophthora root rot (PRR), Phytophthora cinnamomi, is of particular importance to the avocado (Persea americana) industry, which encounters considerable economic losses on account of this pathogen each year. Furthermore, P. cinnamomi is a hemibiotrophic pathogen, suggesting that the SA-signaling pathway plays an essential role in the initial defense response. Therefore, the NPR1 pathway which regulates downstream SA-induced gene expression would be instrumental in defense against P. cinnamomi. Thus, we identified 92 NPR1 pathway-associated orthologs from the P. americana West Indian pure accession genome and interrogated their expression following P. cinnamomi inoculation, using RNA-sequencing data. In total, 64 and 51 NPR1 pathway-associated genes were temporally regulated in the partially resistant (Dusa®) and susceptible (R0.12) P. americana rootstocks, respectively. Furthermore, 42 NPR1 pathway-associated genes were differentially regulated when comparing Dusa® to R0.12. Although this study suggests that SAR was established successfully in both rootstocks, the evidence presented indicated that Dusa® suppressed SA-signaling more effectively following the induction of SAR. Additionally, contrary to Dusa®, data from R0.12 suggested a substantial lack of SA- and NPR1-related defense gene expression during some of the earliest time-points following P. cinnamomi inoculation. This study represents the most comprehensive investigation of the SA-induced, NPR1-dependent pathway in P. americana to date. Lastly, this work provides novel insights into the likely mechanisms governing P. cinnamomi resistance in P. americana.

First Record of Neofusicoccum luteum and Colletotrichum gloeosporioides Causing Anthracnose and Stem End Rot on Avocado Fruits in Greece.

In May 2022, rot symptoms were observed 5 days after storage on fresh avocado fruits cv "Lamb Hass" harvested from a 3.4 ha organic orchard in Chania, Crete exhibiting 30% symptom incidence. Brownish-green sunken lesions and soft rot with dark brown lesions covering up to 50% of the mesocarp on fruits and blackish soft lesions on fruit stem ends were observed. To isolate the pathogens, fruits were surface sterilized using 1% NaOCl for 1 min, placed in 70% ethanol for 30 s and washed twice with sterile distilled water. Then, small pieces were excised from the fruit rot margins and transferred on PDA amended with 0.015% streptomycin-sulfate. Single-spore isolates were incubated on PDA for 10 days and subjected to morphological examination. Two distinct pathogenic fungal isolates were obtained, and their symptoms were re-examined on avocado fruits. The first isolate (A1) obtained from the fruit stem end, initially produced hyaline dense aerial mycelia, being gray and black on the upper and lower surface of the Petri dishes, respectively. The second isolate (A2) obtained from the main body of the fruit, formed round, grayish colonies, with orange conidial aggregates. Based on morphological characteristics (Phillips et al.,2013; Weir et al., 2012), isolates were preliminary identified as Neofusicoccum sp. (A1) and Colletotrichum sp. (A2). Isolates were molecularly identified by sequencing of the ITS-5.8S rRNA, translation elongation factor 1-alpha (tef1) and beta-tubulin (tub2) genes. PCRs were conducted using primer pairs ITS4/ITS5, EF1-728F/EF1986R and Bt2a/Bt2b as well as ITS4/ITS5 and 5'-tef1/3'-tef1 and Bt2a/Bt2b for isolates A1 and A2, respectively (Carbone & Kohn, 1999; Glass & Donaldson, 1995; Rojas et al., 2010; Weir et al., 2012; White et al., 1990). The sequences were deposited into GenBank under the accession numbers OQ852465, OQ867962, OQ867965 for N. luteum and, OQ852466, OQ867963 and OQ867964 for C. gloeosporioides. Based on Multilocus sequence analysis (MLSA), a phylogenetic tree was constructed using concatenated sequences, following Kimura's two parameter model (1980), which confirmed their identity as N luteum and C. gloeosporioides strains. Mature avocado fruits (cv. Hass) were surface sterilized and dried. Consequently, incised fruits were inoculated with mycelial agar plugs (5 mm in diameter) cut from the edge of rapidly growing colonies of N. luteum and C. gloeosporioides strains. Fruits incubated in moist chambers and at 25°C for 5 days in the dark. Fruit bodies and stems were inoculated with the respective isolates and sterile agar plugs in the case of the control. Five fruits were used for each pathogenic trial per fungal isolate, which was repeated twice. After symptom occurrence, these pathogenic isolates were re-isolated successfully and molecularly identified, while exhibiting similar to original symptoms confirming Koch's postulates. While other reports exist on the presence of these pathogens in different countries worldwide, this is the first report of C. gloeosporioides and N. luteum as post-harvest pathogens of avocado, which is an economically important crop of Crete, in Greece (Akgül et al., 2016). This study provides the means for the accurate identification of these fungal pathogens causing avocado fruit rots and taking into consideration the available treatment options can contribute to establishing effective management strategies.

First report of stem rot of Adenium obesum and leaf spot of Persea americana caused by Aspergillus niger in Kazakhstan.

Adenium (Adenium obesum) and avocado (Persea americana) are commonly grown as exotic houseplants in city apartments of Kazakhstan. In April-May 2020, the wilting symptom was observed on the young stems of five 2-year-old A. obesum plants in a city apartment in Saryarqa District, Astana, Kazakhstan (71°25'E, 51°11'N). Leaves turned yellow and then dried up. Plants were completely wilted within 10 days (Fig. 1A). Similar symptoms were observed in newly grown A. obesum plants in November, 2021. At the same time, lesions were found on the leaves of three 3-month-old P. americana plants. Infected leaves displayed dry, dark-brown lesions and fell off easily (Fig. 2A). Both plants were cultivated side by side. The incidence of affected A. obesum was 80% out of 5 plants and P. americana was 100% out of 3 plants. To isolate the causal agent, the infected tissues from different leaves and stems of A. obesum and P. americana plants were cut into small pieces (5 × 5 mm), washed in 70% ethanol for 5 min, and then rinsed three times with sterile distilled water. Cut pieces were placed on potato dextrose agar (PDA) (Laboratorios Conda S.A., Spain) and incubated at 28°C for 7 days. Ten isolates were obtained from leaves and stems of the A. obesum and P. americana symptomatic samples. All fungal colonies were white initially, turned black gradually, reverse side light yellow (Fig. 1B and Fig. 2B), conidiophores biseriate with globose vesicles, conidia were spherical vesicles, light tan to black color, smooth-walled to roughened, and sizes ranged from 3.0 to 3.5 µm (n = 15) (Fig. 1C and Fig. 2C). These observations indicated that all the isolates resembled Aspergillus spp. (Bryan and Fennell 1965). DNA was extracted using the liquid nitrogen and phenol-chloroform extraction method (Butler 2012). A 526 bp product of the ITS region on rDNA and 568 bp product of the calmodulin protein-coding gene was amplified using following primer pairs ITS4/ITS5 (Abliz et al. 2003) and cmd5/cmd6, respectively (Hong et al. 2005). The PCR reaction was done under the following conditions: initial denaturation at 94°C for 5 min, 35 cycles at 95°C for 30 s to denature, 52°C for 40 s for annealing, and 72°C for 50 s for extension. A final extension step at 72°C for 7 min was also included. The sequencing was done using BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and the sequence was deposited in GenBank with accession nos. ON519078 (A. obesum ITS), ON519079 (P. americana ITS), OQ358173 (A. obesum calmodulin) and OQ358174 (P. americana calmodulin). These sequences were compared with other sequences of A. niger in GenBank using BLAST analysis (MG569619.1, MT588793.1, MH478660.1, MZ787576.1 and MW086485.1). The results showed that the sequences of ten isolates were identical and had 98-100% identity with those of Aspergillus niger (Fig. 3). The phylogenetic analysis was carried out with MEGA 11 (Tamura et al. 2021). To confirm the pathogenicity, three asymptomatic plants of each were inoculated with a suspension of conidia via pin-prick inoculation (1.0×106 conidia/ml; obtained from 2-week-old cultures). Control plants were inoculated with sterile distilled water. The inoculated plants were placed in climate chamber (BINDER, Germany) and incubated for 10 days at 28°C. Symptoms were developed in leaves of inoculated plants after 2 days in P. americana and after 5 days in A. obesum. Affected leaves turned yellow and their stems started drying. Symptoms of leaves were similar to those observed on naturally infected plants, while control plants remained asymptomatic. Re-isolation of the pathogen confirmed the presence of the A. niger pathogen. To our knowledge, this is the first report of A. niger causing stem rot of A. obesum and leaf spot of P. americana in Kazakhstan. Since different ornamentals are often planted together in gardens and nurseries, growers should be aware of potential transmission of A. niger among them. This finding provides a foundation to further investigate the biology and epidemiology of this disease so the developmentf diagnostic tools and management measures against it.

Assessment of the Molecular Responses of an Ancient Angiosperm against Atypical Insect Oviposition: The Case of Hass Avocados and the Tephritid Fly Anastrepha ludens.

Anastrepha spp. (Diptera: Tephritidae) infestations cause significant economic losses in commercial fruit production worldwide. However, some plants quickly counteract the insertion of eggs by females by generating neoplasia and hindering eclosion, as is the case for Persea americana Mill., cv. Hass (Hass avocados). We followed a combined transcriptomics/metabolomics approach to identify the molecular mechanisms triggered by Hass avocados to detect and react to the oviposition of the pestiferous Anastrepha ludens (Loew). We evaluated two conditions: fruit damaged using a sterile pin (pin) and fruit oviposited by A. ludens females (ovi). We evaluated both of the conditions in a time course experiment covering five sampling points: without treatment (day 0), 20 min after the treatment (day 1), and days 3, 6, and 9 after the treatment. We identified 288 differentially expressed genes related to the treatments. Oviposition (and possibly bacteria on the eggs' surface) induces a plant hypersensitive response (HR), triggering a chitin receptor, producing an oxidative burst, and synthesizing phytoalexins. We also observed a process of cell wall modification and polyphenols biosynthesis, which could lead to polymerization in the neoplastic tissue surrounding the eggs.

Purification and characterization of avocado (Persea americana) polyphenol oxidase by affinity chromatography.

Polyphenol oxidase (PPO) enzyme was purified from avocado (Persea americana) by ammonium sulfate precipitation 0-80%, dialysis and affinity chromatography. Characterization studies were performed with catechol (0.10 M, pH: 7.2, 37 °C), 4-methyl catechol (0.10 M, pH: 6.0, 37 °C), pyrogallol (0.02 M, pH: 8.5, 5 °C), chlorogenic acid (0.20 M, pH: 6.8, 10 °C) and caffeic acid (0.20 M, pH: 8.5, 10 °C), respectively. Vmax and KM values were determined for catechol (15789.96 U*mL-1*min-1, 10 mM), 4-methyl catechol (6768.40 U*mL-1*min-1, 2 mM), pyrogallol (6802.72 U*mL-1*min-1, 4 mM), chlorogenic acid (1377.97 U*mL-1*min-1, 14.29 mM) and caffeic acid (2567.24 U*mL-1*min-1, 5 mM). PPO was purified as 147.73-fold and 154.00-fold by Sepharose 4B-L-Tyrosine-p-aminobenzoic acid and Sepharose-6B-L-Tyrosine-p-aminobenzoic acid, respectively. 4B isolated PPO gave two bands at 35 and 50 kDa in SDS-PAGE while visible and slightly visible bands at 50-70 kDa and 100 kDa in Native-PAGE. 6B isolated PPO gave bands as distinctively at 50 kDa and unclearly at around 35 kDa in SDS-PAGE while visible and slightly visible bands at 50-70 and 100 kDa in Native-PAGE. The synthesis of original 6B-affinity gel and no any study found in literature on affinity purification of avocado PPO show importance of our study.

Dietary intervention with avocado (Persea americana Mill.) ameliorates intestinal inflammation induced by TNBS in rats.

Nutritional interventions have been shown to be an interesting approach for the treatment of chronic diseases, including inflammatory bowel disease (IBD). Persea americana Mill. (avocado), is a potential food to be used for the prevention or treatment of intestinal inflammation, due to its nutritional value and pharmacological effects. In this study we evaluated if the dietary intervention with avocado fruit pulp could as an intestinal anti-inflammatory diet using a trinitrobenzenesulfonic acid (TNBS) model of intestinal inflammation in rats. For this purpose, 5, 10 or 20% of avocado fruit pulp was incorporated in the diet of rats, for 21 days before and 7 days after TNBS-induced intestinal inflammation. Dietary intervention with avocado fruit pulp (20%) decreased the extension of colonic lesions (1.38 ± 0.99 vs. 2.67 ± 0.76 cm), weight/length colon ratio (151.03 ± 31.45 vs. 197.39 ± 49.48 cm), inhibited myeloperoxidase activity (891.2 ± 243.2 vs 1603 ± 158.2 U/g), reduced tumor necrosis factor-α (53.94 ± 6.45 vs. 114.9 ± 6.21 pg/mg), interleukin-1ß (583.6 ± 106.2 vs. 1259 ± 81.68 pg/mg) and interferon gamma (27.95 ± 2.97 vs. 47.79 ± 3.51 pg/mg) levels and prevented colonic glutathione depletion (2585 ± 77.2 vs 1778 ± 167.2 nmol/g). The consumption of enriched diet with 20% avocado pulp by 28 days did not promote any alterations in the biochemical or behavioral parameters evaluated. Avocado showed intestinal anti-inflammatory activity, modulating immune response, and acting as antioxidant. The dietary intervention with avocado was safe, suggesting its potential as a complementary treatment in intestinal inflammation.

Comprehensive phytochemical characterization of Persea americana Mill. fruit via UPLC/HR-ESI-MS/MS and anti-arthritic evaluation using adjuvant-induced arthritis model.

Persea americana Mill. (avocado fruit) has many health benefits when added to our diet due to various pharmacological activities, such as preventing bone loss and inflammation, modulating immune response and acting as an antioxidant. In the current study, the total ethanol extract (TEE) of the fruit was investigated for in vitro antioxidant and anti-inflammatory activity via DPPH and cyclooxygenase enzyme inhibition. Biological evaluation of the antiarthritic effect of the fruit extract was further investigated in vivo using Complete Freund's Adjuvant (CFA) arthritis model, where the average percentages of body weight change, inhibition of paw edema, basal paw diameter/weight and spleen index were estimated for all animal groups. Inflammatory mediators such as serum IL-6 and TNF-α were also determined, in addition to histopathological examination of the dissected limbs isolated from all experimental animals. Eighty-one metabolites belonging to different chemical classes were detected in the TEE of P. americana fruit via UPLC/HR-ESI-MS/MS. Two classes of lyso-glycerophospholipids; lyso-glycerophosphoethanolamines and lysoglycerophosphocholines were detected for the first time in avocado fruit in the positive mode. The TEE of fruit exhibited significant antioxidant and anti-inflammatory activity in vitro. In vivo anti-arthritic activity of the fruit TEE improved paw parameters, inflammatory mediators and spleen index. Histopathological findings showed marked improvements in the arthritic condition of the excised limbs. Therefore, avocado fruit could be proposed to be a powerful antioxidant and antiarthritic natural product.

First Report of Anthracnose Disease on avocado (Persea americana) caused by Colletotrichum fructicola in China.

In September 2019, anthracnose-like symptoms were observed on fresh avocado fruits cv. "Hass", which were imported from Peru (Mission Produce, Inc.) and purchased at Ganfuyuan store, Guangzhou, China. After being stored for 5 days at room temperature, initial black specks developed into larger brown or black lesions on fruits, and salmon-colored conidial mass in the lesions were observed. To isolate and identify the pathogen, small pieces (5 mm × 5 mm) were excised from the lesion margins of the fruits, which were surface sterilized by 1% NaOCl (1 min), 70% ethanol (30 s) and then washed twice with sterile distilled water (SDW). After sterilization, the tissues were cultured on Potato Dextrose Agar (PDA) to obtain the pure strain NYG. The colonies grown on PDA for 7 days appeared to be cottony, white to pale gray with the presence of a conidial mass. Conidia 11.7-19 × 3.6-6.4 µm (n = 50), hyaline, aseptate, straight and cylindrical with rounded ends, Appressoria 6.2-11.9 × 4.5 × 7.4 µm (n = 50), brown to dark brown in different shapes. Perithecia were thick-walled and globose with a prominent, narrow neck. Asci 31.5-55 × 6-12.5 µm (n = 15), 6-8 spored, clavate to cymbiform. Ascospores 5-18 × 4.5-6 µm (n = 25), hyaline, large guttulate at the centre, slightly curved, rounded ends. Based on the morphological characteristics, the strain NYG was identified as Colletotrichum fructicola (Prihastuti et al. 2009). The identity of the strain was confirmed by means of multi-locus gene sequencing. The genomic DNA was extracted using Ezup Column Fungi Genomic DNA Purification Kit (Sangon Biotech Co., Ltd., China). The internal transcribed spacer (ITS) rDNA region, actin (ACT), chitin synthase (CHS-1), calmodulin (CAL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) partial genes (Templeton et al. 1999, Carbone et al. 1999) were amplified and sequenced, which were deposited in GenBank (OL413493, OL517766, OL517768, OM141126, OL517767). BLASTN analysis revealed that DNA sequences of the isolate showed 100% identity with those of C. fructicola (MT476840.1, MK208862.1, MZ965245.1, JX009665.1, MN982434.1), respectively. A phylogenetic tree analysis based on the concatenated sequences confirmed the isolate as C. fructicola. Pathogenicity was tested by infecting the fresh healthy avocado fruits with the isolated strain NYG. The fruits were surface sterilized, three unwounded and wounded avocados fruits were respectively inoculated with 10 l of conidial suspension (1×106 conidia/ml) by the drop inoculation method. Control fruits were inoculated with SDW containing Tween 20 (1 µl/ml H2O), respectively. All inoculated fruits were incubated at 25°C in the dark. Anthracnose symptoms were observed on the wounded and unwounded fruits after 3 to 5 days post inoculation, respectively. No symptoms were observed in the control on both the wounded and unwounded fruits. The pathogenicity test was performed in duplicate. The inoculated fungus was reisolated from the infected fruits and confirmed as C. fructicola, thus confirming Koch's postulates. C. fructicola represents an important fungal pathogen in several plantations worldwide (Farr et al. 2020), for example, the avocado fruits in Mexico (Dionicio, et al. 2018) and New Zealand (Hofer et al. 2021). This is the first report of anthracnose caused by C. fructicola on imported avocado fruits in China. The results of this study can not only help establish effective quarantine measures against anthracnose disease for imported avocado fruits in China, but also provide important reference to prevent the spread of this disease on China's domestic avocados.

Simultaneous detection of aqueous aluminum(III) and chromium(III) using Persea americana reduced and capped silver nanoparticles.

There is a significant interest to develop sensing devices that detect water toxins, especially heavy metal ions. Although there have already been numerical reports on detecting toxic heavy metal ions, the use of adaptable devices could enable a broader range of sensing applications. Here, we used fresh peel extract (PeA) and dried peel extract (DPeA) of Persea americana (Avocado) as a reducing and capping agent to synthesize and stabilize AgNPs. The dimensions of NPs were controlled by tuning pH, temperature, and volume of the reducing agent. The sensitivity and selectivity of the AgNPs toward various metal ions viz. Ni(II), Cd(II), Al(III), Hg(II), Cr(III), Ba(II), Pb(II), Zn(II), Co(II), Mn(II), Cu(II), Ca(II), Mg(II), and K(I) were studied. The detection probe was found to be selective and sensitive toward Al(III) and Cr(III) ions with the detection limit of 0.04 ppm and 0.05 ppm, respectively. High-resolution transmission electron microscope (HRTEM), ultraviolet-visible (UV-Vis) spectroscopy, and dynamic light scattering (DLS) analysis results confirm an agglomeration-based mechanism for sensing both metal ions. This method can be exploited for the colorimetric detection of toxic heavy metals in real water samples.

This is the first study to report the use of avocado peel extract to synthesize AgNPs in sensing aqueous Al(III) and Cr(III) at trace level concentration.

Physicochemical, Structural, Thermal and Rheological Properties of Flour and Starch Isolated from Avocado Seeds of Landrace and Hass Cultivars.

The objective of this study was to obtain and characterize flours and starches from the avocado seeds of Hass and landrace cultivars. The morphological, physical-chemical, structural, thermal and rheological characteristics were evaluated. The flour yield of the Hass and landrace cultivars was 41.56 to 46.86% (w/w), while for starch, it was 35.47 to 39.57% (w/w) (cv. Hass and landrace, respectively). Scanning electron microscopy (SEM) revealed the presence of oval starch granules and other particles in flour, in contrast to flours, starches showed lower ash, proteins and lipids content. However, the amylose content was higher in starches (42.25-48.2%). Flours showed a higher gelatinization temperature (Tp = 73.17-73.62 °C), and their starches presented greater gelatinization enthalpy (∆Hgel = 11.82-13.43 J/g). All samples showed a B-type diffraction pattern, and the crystallinity was higher in the flours. The rheological analysis (flow curves and viscoelastic tests) evidenced a pseudoplastic (n = 0.28-0.36) behavior in all samples analyzed, but the consistency index (k) was higher in starches. In general, the flours and starches from avocado seeds presented interesting proximal, thermal and functional properties for possible application in food systems, and these findings could contribute to the revaluation of this by-product.

Nephroprotective and anti-inflammatory potential of aqueous extract from Persea americana seeds against cadmium-induced nephrotoxicity in Wistar rats.

Cadmium is a toxic metal and poses a high environmental risk to animals and humans, alike. It is thus pertinent to search for medicinal plants in protecting against cadmium toxicity. This study aims at investigating the ability of aqueous extract of Persea americana seeds (AEPA) in ameliorating the toxic effects of cadmium in the kidneys of cadmium-exposed Wistar rats. Male Wistar rats were grouped into five, of six animals each. Different groups of animals received normal saline (control group), 200 mg/kg body weight AEPA, 400 mg/kg AEPA, and standard drug, Livolin Forte, respectively. A last group of animals was left untreated. To induce toxicity, all animals, except the control group, were exposed to cadmium (200 mg/L, as CdCl2) in their main drinking water for 21 days. Biochemical analysis of serum kidney markers, oxidative stress and antioxidant status, as well as anti-inflammatory activities, was done using standard methods and kits. In silico analysis was performed on phytochemicals reported to be abundant in AEPA. Treatment with 400 mg/kg AEPA significantly reversed (P ≤ 0.05) the adverse effect of cadmium on serum creatinine, urea, uric acid and blood urea nitrogen, and restored (P ≤ 0.05) antioxidant status, evidenced by its significant effect on superoxide dismutase, catalase, glutathione-S-transferase, glutathione peroxidase, reduced glutathione, and lipid peroxidation activities. AEPA, at 400 mg/kg also exhibited significant anti-inflammatory effects, which was shown by reduced interleukin-2 and tumour necrosis factor α activities. Molecular docking of phytochemicals with the selected protein target also confirmed the therapeutic potential of AEPA. The study concluded that aqueous extract of AEPA protects against cadmium-induced kidney toxicity and inflammation.

γ-Lactones from Persea americana and Persea fulva - in Vitro and in Silico Evaluation of Trypanosoma cruzi Activity.

In the present study, five known γ-lactones (majoranolide B - 1, majorenolide - 2, majorynolide - 3, lincomolide D - 4, and isolinderanolide E - 5), as well as a new one (perseanolide - 6), were isolated from Persea fulva and P. americana. All isolated compounds exhibited potential activity against trypomastigote forms of Trypanosoma cruzi, whereas compounds 2 (EC50 of 4.8 µM) and 6 (EC50 of 3.6 µM) displayed superior activity than the positive control benznidazole (EC50 of 16.4 µM), with selectivity index (SI) values of 17.8 and >55.6, respectively (benznidazole, SI>12.2). Molecular docking studies were performed for 1-6 against six T. cruzi molecular targets. Using this approach, we observed that, even though perseanolide (6) showed favorable docking to several studied targets, the results were especially promising for hypoxanthine phosphoribosyl transferase (PDB 1TC1). As PDB 1TC1 is associated to the transference of a monophosphorylated ribose from phosphoribosylpyrophosphate (PRPP) in the ribonucleotide synthesis pathway, this interaction may affect the survival of T. cruzi in mammalian cells. The data herein also indicate that possible intermolecular interactions between 6 and PDB 1TC1 derive from (i) hydrogen bonds in the α,ß-unsaturated-γ-lactone unity and (ii) hydrophobic interactions in the long-chain alkyl group. Based on our results, perseanolide (6), reported for the first time in this work, can auspiciously contribute to future works regarding new trypanocidal agents.

First report of Colletotrichum fructicola, C. perseae, and C. siamense causing anthracnose disease of avocado (Persea americana) in New Zealand.

In January and March 2019, an inspection of 11 commercial 'Hass' avocado orchards in mid-North and Tauranga (New Zealand) was conducted by NZ Avocado Growers Association Inc. (NZAGA) and the samples were sent to Plant Diagnostics Limited for investigation of a newly observed fruit staining symptom termed "tannin stain". Fruit symptoms consisted of areas of minute small spots which coalesced into areas of tear staining associated with water movement over the fruit's surface (Supplementary Fig. 1). Up to seven trees per orchard were sampled targeting symptomatic fruit with the aim of determining the cause of the problem. Fruit was surface disinfected for 4 minutes in 1% sodium hypochlorite solution and sections from lesions were plated on agar medium (prune extract agar) to isolate any plant pathogens. The predominant fungi isolated, represented species in the Colletotrichum acutatum, C. gloeosporioides, and C. boninense species complexes. Since the morphological characters within these complexes overlap (see Supplementary Fig. 2 for examples), the isolates were differentiated by amplification and sequencing of the glyceraldehyde-3-phosphate dehydrogenase (GPDH) gene and, where necessary, the calmodulin (CAL) gene and/or the Apn2-Mat1-2 intergenic spacer region (ApMat) locus (Weir et al., 2012; Rojas et al., 2010). The sequence analysis revealed eight Colletotrichum species comprising C. alienum, C. aotearoa, C. cigarro, C. fioriniae, C. fructicola, C. karstii, C. perseae, and C. siamense. This range included three species that have not previously been recorded in New Zealand: C. fructicola (Cf), C. perseae (Cp), and C. siamense (Cs). Colonies for all these three fungi were white to grey with salmon-coloured and black acervuli. Conidia were aseptate, hyaline, straight, cylindrical, with broadly rounded ends, forming on cylindrical conidiogenous cells. The respective GPDH, CAL, and/or ApMat sequences of the Cf, Cp, and Cs isolates were identical to reference sequences of representative isolates in GenBank (e.g. ApMat: Cf - KX620181, Cp - KX620177, Cs - KP703788). An isolate for each species is stored in the International Collection of Microorganisms from Plants (Cf - ICMP22409, Cp - ICMP22431, Cs - ICMP22411) and sequences are deposited in GenBank (accession numbers MT522858-MT522865). Pathogenicity of each of the newly recorded species was confirmed on freshly picked 'Hass' avocado fruit. After surface disinfection with 1% sodium hypochlorite solution for 5 minutes, fruit was triple washed with sterile water and air dried. Five fruits per species were pin-pricked and inoculated with 10µL of conidial suspension (7 x 106 to 1 x 107 conidia/mL) prepared with sterile water containing Tween 20 (1µL/mL H2O) from 6-day-old cultures grown on PDA. Control fruit was pin-pricked and mock-inoculated with sterile water containing Tween 20 (1µL/mL H2O). All fruit was incubated in moist chambers at 25°C for 7 days. The three Colletotrichum species produced anthracnose symptoms on inoculated fruit whereas no symptoms were observed on control fruit (Supplementary Fig. 3). Each one of the species was successfully re-isolated from symptomatic tissue and identified using the methods described above, fulfilling Koch's postulates. While Cf and Cs have been reported from several hosts and countries to date (Weir et al. 2012), Cp has only been found from avocado in Israel (Sharma et al. 2017) and grape in Japan (Yokosawa et al. 2020). Although a number of species from the C. gloeosporioides species complex, i.e. C. alienum, C. aotearoa, C. cigarro, and C. gloeosporioides have been previously associated with avocado diseases in New Zealand, the detections of Cf, Cp, and Cs represent first records. In this study, eight Colletotrichum species were associated with the "tannin stain" fruit symptoms in New Zealand avocado orchards. The individual contribution of the newly recorded pathogens Cf, Cp, and Cs to the observed disease symptoms was not determined, but their detection highlights the importance of sequence-based identification of Colletotrichum species, as morphology is insufficiently robust to separate cryptic species. Accurate identification of pathogens provides knowledge of species biodiversity that may be useful in biosecurity decision making. Since it has been reported that fungicide treatment efficiencies differ for some closely related Colletotrichum species on grape (Yokosawa et al. 2020), accurate identification might also contribute to establishing effective management strategies.