First Report of Diaporthe hongkongensis Causing Leaf Blight on Macadamia in China.

Macadamia (Macadamia ternifolia Maiden and Betche) belongs to the Proteaceae family (Li et al. 2022). In the hilly areas of Guangxi (southern China), macadamia trees are an important source of revenue. The planting area in Guangxi has increased in recent years, exceeding 53,333 hectares by the end of 2022, but this increase is also associated with emergency of, macadamia diseases. Leaf blight symptoms were observed in 37/241 macadamia trees (15% incidence) in a plantation in Nanning, Guangxi province in China, during June, 2022. Disease severity on infected trees ranged from 5% to 60%. The disease developed from the tips or margins of leaves, causing the leaves to turn brown, and later gradually withered (Fig. 1 A). Ten leaves with lesions were collected from five macadamia trees (two leaves per tree. Thereafter, small segments (3 to 4 mm²) excised from the margins of ten lesions were surface sterilized in 75% ethanol for 30 s and 1% hypochlorite for 90 s and Page 1 of 6 2 rinsed in sterile water, before plating onto potato dextrose agar (PDA) medium. Plates were incubated under lighting during the daytime, and darkness at night-time for 5 days at 25℃. Twenty-two purified colonies were generated by subculturing hyphal tips, of which eight exhibited similar morphology and were further characterized. The colonies on PDA were gray with a white outer ring and flat lawn on the surface (Fig. 1 B). The pycnidia were superficial to semi-immersed on PDA, solitary to aggregated, globose to sub-globose, brown to black and oozed yellow mucilaginous masses (Fig.1 C). The α-conidia were unicellular, hyaline elliptical or fusiform, and measuring 4-8 × 1.9-4 µm (n=30) ， whereas the ß-conidia were hyaline, long, straight or curved, measuring 20-23 × 0.9-2 µm (n=30) (Fig. 1 D-E). The morphological features were similar to Diaporthe hongkongensis (Dissanayake et al. 2015). The eight morphologically similar isolates were identified as D. hongkongensis using the internal transcribed spacer (ITS) region, but only one isolate, JG11, was selected for further molecular identification. Five target genes, including the ITS region, translation elongation factor 1 alpha (EF1-α), beta-tubulin genes (TUB2), calmodulin (CAL), and histone H3 (HIS) were amplified and sequenced using primers ITS1/ITS4, EF1-728F/EF1-986R, Bt2a/Bt2b, CAL-228F/CAL-737R, and CYLH3F/H3-1b, respectively (Carbone and Kohn 1999). The sequences were deposited in GenBank under accession numbers OQ932790 (ITS) and OR147955-58 for EF1-α, TUB, CAL and HIS genes, respectively. BLAST search of GenBank showed that ITS, EF1-α, TUB, CAL, and HIS sequences of JG11 were similar to Page 2 of 6 3 those of D. hongkongensis NR111848 (99.22% identity), KY433566 (99.72%), MW208603 (99.42%), MW221740 (99.80%), and MW221661 (99.79%), respectively. Phylogenetic analysis of concatenated sequences was performed with IQ-TREE software. JG11 was grouped in the same clade as other Diaporthe hongkongensis isolates (Fig. 2). Pathogenicity experiments were carried out on healthy macadamia trees in a greenhouse. Three macadamia trees were used as negative controls where five uninjured leaves per tree were sprayed with sterile distilled water. Uninjured five leaves per tree of three other macadamia trees were sprayed with conidia suspension of the isolate JG11 at a concentration of 1×106. Each treatment was repeated 3 times independently, with 5 leaves per tree (Liu et al. 2023; Havill et al. 2023; Zhang et al. 2022). Plastic bags were placed over all inoculated leaves. The average daily temperature and relative humidity in the greenhouse were 32°C and 65%, respectively. Two days later, browning appeared on the leaves inoculated with the spore suspension and expanded outward. After 5 days, all macadamia leaves inoculated with the fungal spores began to wither, while controls remained asymptomatic (Fig. 1 H-I). D. hongkongensis was consistently re-isolated and purified from inoculated leaves and the identity was confirmed by morphological identification and molecular analysis, completed Koch's postulates. D. hongkongensis has been reported on peach (Zhang et al. 2021), grapevine trunk (Dissanayake et al. 2015) and Cunninghamia lanceolata (Liao et al. 2022). To our knowledge, this is the first report of D. hongkongensis causing leaf blight on macadamia in China. These findings provide a foundation for future research on the epidemiology and control of this newly emerging disease of macadamia.

Comparative Transcriptome Analysis Reveals Key Functions of MiMYB Gene Family in Macadamia Nut Pericarp Formation.

Macadamia nuts are one of the most important economic food items in the world. Pericarp thickness and flavonoid composition are the key quality traits of Macadamia nuts, but the underlying mechanism of pericarp formation is still unknown. In this study, three varieties with significantly different pericarp thicknesses, namely, A38, Guire No.1, and HAES 900, at the same stage of maturity, were used for transcriptome analysis, and the results showed that there were significant differences in their gene expression profile. A total of 3837 new genes were discovered, of which 1532 were functionally annotated. The GO, COG, and KEGG analysis showed that the main categories in which there were significant differences were flavonoid biosynthesis, phenylpropanoid biosynthesis, and the cutin, suberine, and wax biosynthesis pathways. Furthermore, 63 MiMYB transcription factors were identified, and 56 R2R3-MYB transcription factors were clustered into different subgroups compared with those in Arabidopsis R2R3-MYB. Among them, the S4, S6, and S7 subgroups were involved in flavonoid biosynthesis and pericarp formation. A total of 14 MiMYBs' gene expression were verified by RT-qPCR analysis. These results provide fundamental knowledge of the pericarp formation regulatory mechanism in macadamia nuts.

MassARRAY and SABER Analyses of SNPs in Embryo DNA Reveal the Abscission of Self-Fertilised Progeny during Fruit Development of Macadamia (Macadamia integrifolia Maiden & Betche).

Yield in many crops is affected by abscission during the early stages of fruitlet development. The reasons for fruitlet abscission are often unclear but they may include genetic factors because, in some crops, self-pollinated fruitlets are more likely to abscise than cross-pollinated fruitlets. Pollen parentage can also affect final fruit size and fruit quality. Here, we aimed to understand the effects of pollen parentage on fruitlet retention and nut quality in orchards of macadamia (Macadamia integrifolia Maiden & Betche). We identified the pollen parent of macadamia 'cultivar '816' embryos by analysing single nucleotide polymorphisms (SNPs) in their DNA using customised MassARRAY and Single Allele Base Extension Reaction (SABER) methods. This allowed us to determine the proportions of self-fertilised and cross-fertilised progeny during premature fruit drop at 6 weeks and 10 weeks after peak anthesis, as well as at nut maturity. We determined how pollen parentage affected nut-in-shell (NIS) mass, kernel mass, kernel recovery, and oil concentration. Macadamia trees retained cross-fertilised fruitlets rather than self-fertilised fruitlets. The percentage of progeny that were cross-fertilised increased from 6% at 6 weeks after peak anthesis to 97% at nut maturity, with each tree producing on average 22 self-fertilised nuts and 881 cross-fertilised nuts. Three of the four cross-pollen parents provided fruit with significantly higher NIS mass, kernel mass, or kernel recovery than the few remaining self-fertilised fruit. Fruit that were cross-fertilised by '842', 'A4', or 'A203' had 16-29% higher NIS mass and 24-44% higher kernel mass than self-fertilised fruit. Nuts that were cross-fertilised by 'A4' or 'A203' also had 5% or 6% higher kernel recovery, worth approximately $US460-540 more per ton for growers than self-fertilised nuts. The highly selective abscission of self-fertilised fruitlets and the lower nut quality of self-fertilised fruit highlight the critical importance of cross-pollination for macadamia productivity.

Effect of dietary supplementation of macadamia oil on the growth, immune function, physio-biochemical components and thyroid activity of growing rabbits.

The current research aimed to assess the effects of dietary macadamia oil (MO) on carcass traits, growth performance, physio-biochemical components, immune function, thyroid hormones and inflammation markers of growing rabbits. A total of 96 growing rabbits were randomly distributed into four treatments, with 24 rabbits in each group. The rabbits were fed a basal diet (control group) or a diet supplemented with MO at 0.5 (MO0.5), 1 (MO1.0) and 2 (MO2.0) mL/kg of diet for eight weeks. The daily body weight gain and feed conversion ratio showed a quadratic improvement with increasing levels of MO, and the optimal dose was 1.25 mL/kg of diet. Increasing levels of MO also had a quadratic effect on hepatic and renal functions. Dose-response curves revealed that the optimal doses of MO were 1.50, 1.75 and 1.25 mL/kg of diet for total bilirubin, gamma-glutamyl transferase, and creatinine respectively. A quadratic relationship was observed between the increased levels of MO and tumour necrosis factor-α (p = 0.038), interleukin-6 (p = 0.014) and immunoglobulins (p = 0.016 and IgM p = 0.026). Additionally, a linear relationship was observed between the increment in MO levels and both nitric oxide (p = 0.040) and interleukin-4 (p = 0.001). The activities of superoxide dismutase and glutathione peroxidase showed a linear increase with increasing dietary MO content, while xanthine oxidase showed a linear decrease. Total antioxidant capacity showed quadratic improvement (p = 0.035) with the dietary treatment, with the optimal dose observed at 1.25 mL/kg of diet. The inclusion of MO in the diet had a linear effect on the activity of thyroxine (p = 0.001). Therefore, supplementation of MO at a dose of 1 or 1.5 mL/kg of diet in growing rabbits' diets can improve growth and carcass traits, sustain thyroid function by supporting immunity, and reduce oxidative/inflammation pathways.

De novo chromosome level assembly of a plant genome from long read sequence data.

Recent advances in the sequencing and assembly of plant genomes have allowed the generation of genomes with increasing contiguity and sequence accuracy. Chromosome level genome assemblies using sequence contigs generated from long read sequencing have involved the use of proximity analysis (Hi-C) or traditional genetic maps to guide the placement of sequence contigs within chromosomes. The development of highly accurate long reads by repeated sequencing of circularized DNA (HiFi; PacBio) has greatly increased the size of contigs. We now report the use of HiFiasm to assemble the genome of Macadamia jansenii, a genome that has been used as a model to test sequencing and assembly. This achieved almost complete chromosome level assembly from the sequence data alone without the need for higher level chromosome map information. Eight of the 14 chromosomes were represented by a single large contig (six with telomere repeats at both ends) and the other six assembled from two to four main contigs. The small number of chromosome breaks appears to be the result of highly repetitive regions including ribosomal genes that cannot be assembled by these approaches. De novo assembly of near complete chromosome level plant genomes now appears possible using these sequencing and assembly tools. Further targeted strategies might allow these remaining gaps to be closed.

Evaluation of In-Field Inoculation Methods for Characterization of Macadamia Germplasm to Husk Spot Incidence and Severity.

Husk spot, a fungal disease of macadamia pericarps (Pseudocercospora macadamiae), induces premature abscission in several major commercial cultivars. Breeding for resistance to husk spot is a priority of the Australian macadamia industry. Due to the large tree size of macadamia and high numbers of progeny in breeding populations, inoculating for resistance screening is laborious and time consuming. Previously utilized methods included direct applications of P. macadamiae suspensions and the hanging of bags of diseased husks above developing fruit in tree canopies. In this study, both methods were modified to allow for efficient application in large-scale breeding populations, and their efficacy was evaluated. Two quantities of diseased husk per bag, 'large' (75 g) and 'small' (30 g), and two concentrations of sprayed P. macadamiae suspensions, 'stock' (5 × 105 propagules/ml) and 'dilute' (2.5 × 105 propagules/ml), were tested across two fruiting seasons. Treatments were compared against a control (sterile water) in commercial cultivars A38 and A4. Husk spot incidence and severity produced by small bags were significantly affected by season. A significant season effect was less common for other treatments. All four treatments infected over 50% of target fruit in each season, but the highest husk spot incidence across both seasons (≥85%) was produced from large bags. Overall, the large bags were the most reliable method for infection of target fruit. Results also demonstrate the importance of considering the effect of season when selecting husk spot inoculation methods.

First report of Phytopythium vexans causing root rot on Macadamia integrifolia in China.

Soil-borne plant-pathogenic Phytopythium spp. can cause root rot and damping off on important plant species, resulting in serious economic loss. A survey in October 2021 identified soil-borne diseases occurring on Macadamia integrifolia in Yunnan Province, China. Microbes were isolated from necrotic roots of 23 trees with root rot symptoms by growing on cornmeal-based oomycete-selective 3P (Haas 1964) and P5APR (Jeffers and Martin, 1986) media at 24ºC in the dark for 7 days. Of the 56 single-hyphal isolates obtained, 18 were morphologically similar to Phytopythium vexans (van der Plaats-Niterink 1981; de Cock et al. 2015). Isolates LC04 and LC051 were selected for molecular analyses. The internal transcribed spacer (ITS) region and the cytochrome c oxidase subunit II (CoxII) gene were PCR-amplified using universal primers ITS1/ITS4 (White et al. 1990) and oomycete-specific primers Cox2-F/Cox2-RC4 (Choi et al. 2015), respectively. The PCR products were sequenced with the amplification primers and sequences were lodged in Genbank (Accession no. OM346742, OM415989 for ITS, OM453644, OM453643 for CoxII for isolates LC04 and LC051, respectively). The top BLAST hit in the Genbank nr database for all four sequences was Phytopythium vexans (>99% identity). A maximum-likelihood phylogenetic tree was constructed with analogous concatenated ITS and CoxII sequences from either type or voucher specimens of 13 Phytopythium species in the same phylogenetic clade as P. vexans (Table 1; Bala et. al 2010). Isolates LC04 and LC051 grouped most closely to P. vexans, with LC051 basal and sister to LC04 and P. vexans voucher specimen CBS119.80 with 100% support (Fig. 1). Millet seed inoculated with agar pieces colonized by P. vexans LC04 and LC51 was used to fulfill Koch's postulates (Li et al. 2015) in a completely randomized experimental design. Four 6-month-old M. integrifolia var. Keaau (660) seedlings were transplanted into pasteurized commercial potting mix containing 0.5% (w/w) inoculum. Plants were grown in free draining pots and watered once a day. At 14 days post-inoculation, roots were discolored compared to control plants inoculated with millet seed mixed with agar plugs lacking P. vexans (Fig. 2). By 30 days post-inoculation, infected roots were discolored with obvious decay and reduction in root system size. Control plants were symptomless. P. vexans was successfully re-isolated from two lesioned roots from each plant. The infection experiment was done twice, demonstrating that P. vexans LC04 and LC51 caused root disease on M. integrifolia. P. vexans causes root rot, damping-off, crown rot, stem rot or patch canker on economically important trees in many parts of the world, including seven plant species in China (Farr and Rossman 2022). This is the first report of pathogenic P. vexans on M. integrifolia in China. Reports of pathogenic P. vexans on multiple hosts in several parts of the world suggest it should be considered a quarantine risk and included in risk mitigation or pest management plans that include other species of Phytopythium, or species of Pythium or Phytophthora, to which P. vexans has many similarities (de Cock et al. 2015).

Macadamia nut-specific IgE levels for predicting anaphylaxis.

BACKGROUND: Despite the high risk of anaphylaxis in patients with a macadamia nut allergy (MdA), little is known about the significance of macadamia nut-specific immunoglobulin E (Md-sIgE). Thus, this study aimed to investigate the utility of Md-sIgE for predicting anaphylaxis. METHODS: Children with suspected MdA who visited our hospital were included. MdA was defined as either failing the 3-g macadamia nut (Md) oral food challenge (OFC) or confirming obvious immediate symptoms following Md ingestion. Non-MdA was defined as passing the 3-g Md OFC. RESULTS: A total of 41 children (29 [71%] males) with a median age of 7.7 years were included. The median Md-sIgE level was 2.23 kUA /L. Among the 21 children diagnosed with MdA, eight and 13 children did (An group) and did not (non-An group) develop anaphylaxis. Twenty children were included in the non-MdA group. The Md-sIgE level was significantly higher in the An group relative to the others (7.97 vs. 1.92 kUA /L, p < .001). Furthermore, the Md-sIgE level was significantly higher in the An group than in the non-An group (7.97 vs. 1.92 kUA /L, p = .02). However, there was no significant difference in the Md-sIgE between the non-An and non-MdA groups (1.92 vs. 1.90 kUA /L, p > .99). The area under the curve for predicting anaphylaxis in Md-sIgE was 0.92 (95% CI: 0.83-1.00), and the optimal cut-off value was 3.76 kUA /L. CONCLUSION: Md-sIgE levels were useful in predicting anaphylaxis. Above the cut-off value, we emphasize paying careful attention to the risk of anaphylaxis.

Pollen limitation and xenia effects in a cultivated mass-flowering tree, Macadamia integrifolia (Proteaceae).

BACKGROUND AND AIMS: Pollen limitation is most prevalent among bee-pollinated plants, self-incompatible plants and tropical plants. However, we have very little understanding of the extent to which pollen limitation affects fruit set in mass-flowering trees despite tree crops accounting for at least 600 million tons of the 9200 million tons of annual global food production. METHODS: We determined the extent of pollen limitation in a bee-pollinated, partially self-incompatible, subtropical tree by hand cross-pollinating the majority of flowers on mass-flowering macadamia (Macadamia integrifolia) trees that produce about 200 000-400 000 flowers. We measured tree yield and kernel quality and estimated final fruit set. We genotyped individual kernels by MassARRAY to determine levels of outcrossing in orchards and assess paternity effects on nut quality. KEY RESULTS: Macadamia trees were pollen-limited. Supplementary cross-pollination increased nut-in-shell yield, kernel yield and fruit set by as much as 97, 109 and 92 %, respectively. The extent of pollen limitation depended upon the proximity of experimental trees to trees of another cultivar because macadamia trees were highly outcrossing. Between 84 and 100 % of fruit arose from cross-pollination, even at 200 m (25 rows) from orchard blocks of another cultivar. Large variations in nut-in-shell mass, kernel mass, kernel recovery and kernel oil concentration were related to differences in fruit paternity, including between self-pollinated and cross-pollinated fruit, thus demonstrating pollen-parent effects on fruit quality (i.e. xenia). CONCLUSIONS: This study is the first to demonstrate pollen limitation in a mass-flowering tree. Improved pollination led to increased kernel yield of 0.31-0.59 tons ha-1, which equates currently to higher farm-gate income of approximately $US3720-$US7080 ha-1. The heavy reliance of macadamia flowers on cross-pollination and the strong xenia effects on kernel mass demonstrate the high value that pollination services can provide to food production.

First Report of Colletotrichum fructicola Causing anthracnose on macadamia in China.

Macadamia (Macadamia ternifolia Maiden and Betche) is an important commercial crop in the world and has the reputation of being the king of nuts (Liu et al. 2019). In August 2020, symptoms of anthracnose appeared on leaves of macadamia in Chongzuo, Guangxi Province, China, with an incidence of 15-20%. The disease developed from the edge of leaf. Initially, the disease symptoms on leaves were faded green spots, light yellow. After expanding and linking together, the leaves appeared brown or black irregular spots, and the edges of diseased leaves dried up and formed large necrosis, eventually leading to defoliation. A large number of orange-yellow spots (acervuli) developed on the diseased parts. Under high humidity conditions, the diseased part was grayish-brown or black, and a large number of yellowish-brown conidia were produced on the leaf surface (Fig.1 A-E). Ten symptomatic leaves were collected and washed with distilled water. Twelve lesion marginal tissues were sterilized with 75% ethanol (V/V) for 30 s and 1% NaOCl for 1min and rinsed with sterile distilled water, plated on potato dextrose agar (PDA) and incubated at 28°C under light. After 3 days, the incubated samples all produced similar cultural morphology. One isolate named GXMC2 as a representative was selected for following study. The colony by single-spore purification on PDA were grayish green with a white outer ring and cottony on surface, pale blackish green in reverse side (Fig.1 F). Conidia with oil droplets were solitary, cylindrical, transparent and measuring 13.78 to 19.25 µm (average 16.90 µm) × 5.14 to 7.33 µm (average 6.23 µm) (n=100) (Fig.1 G). Appressoria were brown to dark brown, with different shapes such as ovoid, elliptical or irregular, some with lobes. The average size was 7.89 to 13.25 µm (average 10.64 µm) × 5.76 to 9.02 µm (average 7.86 µm) (n=100) (Fig.1 H). No setae were found. The isolate was identified as Colletotrichum fructicola on the basis of the morphology of the colonies, conidia and appressoria (Park et al. 2018). The six target gene sequences, including internal transcribed spacer (ITS), ß-tubulin (TUB), actin (ACT), histone3 (HIS3), chitin synthase A (CHS), and glyceraldehydes-3-phosphate dehydrogenase (GAPHD) (Qiu et al. 2020), were selected for PCR amplification. The resulting sequences were deposited in GenBank under accession numbers MZ821661, MZ821660, MZ821662, MZ821663, MZ821664 and MZ821665 respectively. Phylogenetic analysis of the concatenated sequences were performed with MEGA 7.0 software. The isolate was grouped in the same clade as other C. fructicola (Fig.2). In May 2022, Inoculation was conducted in the field. Four-year-old macadamia leaves were disinfected with 75% alcohol. The conidial suspension was sprayed on 5 unwounded healthy leaves, and 5 leaves sprayed with sterile distilled water served as control. The experiment was replicated 3 times, with each replicate containing 5 leaves. The average daily temperature and average daily relative humidity in the field were 30°C and 62%, respectively. After 2 days, yellow-brown spots appeared on the inoculated leaves and expanded outward. After 4 days, the diseased areas were dark brown, and the controls remained asymptomatic. The same fungal pathogen was reisolated and purified from inoculated leaves and the identity was confirmed by morphological characterization and molecular analysis, confirming Koch's postulates (Fig.1 I-J). In China, C. fructicola has been reported on Passiflora edulis Sims, Brassica parachinensis, Illicium verum, Peucedanum praeruptorum, etc. (Li et al. 2021; Yu et al. 2022; Zhao et al. 2021; Ma et al. 2020). To our knowledge, this is the first report of anthracnose of macadamia caused by C. fructicola in China. This study provides the basis for further research on this disease.

First report of Neofusicoccum parvum causing leaf spot disease on Macadamia integrifolia in China.

The macadamia industry is developing rapidly in China. A brown leaf spot disease was noted in six Macadamia integrifolia plantations in Lincang, Yunnan, in October 2021. Over 60% of trees sampled had brown leaf spot symptoms, among approx.15,000 trees planted in these areas. Lesions (3 to 5 mm dia.) were small round brown spots with yellow edges. Lesions on severely infected leaves were darker and larger, with irregular shape (8 to 10 mm long, 3 to 6 mm wide). About 10% of diseased leaves had lesions characterized by a shot hole surrounded by a yellow halo. Potential pathogens were isolated from four randomly-selected symptomatic leaves from each of the six plantations by cutting lesion edges into small pieces. The pieces were surface sterilized, placed onto water agar containing 100 ppm aureomycin and incubated for 5 days at 24°C in the dark. Subculturing microbial growth on potato dextrose agar produced single-hyphal isolates with white fluffy aerial mycelia that turned pale olivaceous gray after 4 to 5 days. In four randomly-selected cultures, conidia were single celled, hyaline, spindle shaped to oval, and measured 10.9 to 16.3 µm long and 4.0 to 6.2 µm wide (n = 50). These characteristics matched those of Neofusicoccum parvum (Pavlic et al. 2009). Isolate LC013 was randomly selected as a representative individual for molecular identification. Internal transcribed spacer (ITS; ITS1/ITS4 primers; White et al. 1990), beta-tubulin gene (tub2; BT2A/BT2B primers; Glass and Donaldson 1995) and translation elongation factor 1-alpha gene (tef1-α; EF1-728F/EF2 primers; Carbone and Kohn 1999; O'Donnell et al. 1998) regions were PCR amplified from genomic DNA. Sequences of the products were used to BLAST probe the type specimen nucleotide sequences in GenBank. The LC013 sequences (GenBank accessions OM392021 (ITS); OM453641 (tub2); OM567656 (tef1-α)) had >99% sequence identity with analogous sequences from the type specimen of N. parvum CBS 138823 (accessions AY236943 (ITS); AY236917 (tub2); AY236888 (tef1-α)). Isolate LC013 was sister to N. parvum type strain in a maximum-likelihood (ML) tree constructed from analogous concatenated ITS, tef1-α, and tub2 sequences of 27 species that are phylogenetically closely-related to LC013 based on the ITS single locus ML tree. Koch's postulates were tested twice with two isolates by wounding leaves of four 14-month-old M. integrifolia seedlings with a sterile needle and placing a 5-mm-diameter agar plug containing N. parvum on the wound site. PDA plugs alone were used as uninoculated controls. Leaves were covered with sealed bags to maintain >90% humidity for 24 hours. All plants were kept in the same glasshouse under natural conditions. Leaves of inoculated plants began to discolor at 5 days post-inoculation (dpi). Brown spot symptoms were observed at 9 dpi. Control plants were symptomless. N. parvum was re-isolated from leaf lesions of the infected plants, but not from control plants, thus fulfilling Koch's postulates. N. parvum is an aggressive pathogen that causes severe disease on important tree and woody species, including M. integrifolia (Liddle et al. 2019). In China, it has been reported to cause leaf spot disease on 26 plant species (Farr and Rossman 2022), but this is the first report of N. parvum causing leaf spot disease on M. integrifolia. Further investigation is required to estimate the importance of this pathogen to the macadamia industry in China.

Screening for Susceptibility of Macadamia to Euwallacea fornicatus and its Fungal Symbiont Fusarium euwallaceae.

The polyphagous shothole borer (Euwallacea fornicatus, PSHB), an ambrosia beetle, with its fungal symbiont, Fusarium euwallaceae, is responsible for Fusarium dieback (FD) in a wide range of woody hosts. In 2019, the first suspected case of E. fornicatus was reported in macadamia in South Africa. The aims of this study were to confirm the E. fornicatus report and thereafter to assess the susceptibility of commercially planted macadamia cultivars to FD caused by F. euwallaceae. The identities of the beetle and associated fungal symbionts were confirmed by means of DNA sequence analysis of the 28S ribosomal large subunit gene for beetles and the internal transcribed spacer region for fungi. Isolates identified as Fusarium species were further characterized by phylogenetic analysis of the translation elongation factor 1α and the ß-tubulin gene regions. Thereafter, Koch's postulates regarding F. euwallaceae were fulfilled on a mature Macadamia integrifolia tree planted at the experimental farm of the University of Pretoria. In order to determine susceptibility against FD, additional cultivar screening was conducted on nine commercially planted cultivars by means of pathogenicity trials using sterilized or inoculated toothpicks inserted into detached branches. Detached branch inoculations showed no significant lesion development six weeks post inoculation, except for cultivar 816. The restricted growth of F. euwallaceae observed in macadamia tissues therefore suggests that macadamia may not be a suitable host for F. euwallaceae and that the threat of FD in macadamia in the event of E. fornicatus infestation is less than for other E. fornicatus hosts. Future work on beetle attraction to macadamia is recommended for a more comprehensive understanding of the interaction between E. fornicatus and its fungal symbionts and macadamia.

Response of the macadamia seed weevil Kuschelorhynchus macadamiae (Coleoptera: Curculionidae) to Metarhizium anisopliae and Beauveria bassiana in laboratory bioassays.

Macadamia seed weevil, Kuschelorhynchus macadamiae Jennings and Oberprieler, is a major pest of macadamia in eastern Australia, causing yield losses of up to 15%. Current control methods involve two applications of acephate per season but more recently have moved to a single application of indoxacarb, combined with the collection and destruction of fallen nuts that contain developing larvae. As a first step towards reducing the dependence of the industry on synthetic insecticides, we tested six isolates of M. anisopliae, six isolates of B. bassiana and one commercial B. bassiana product (Velifer® biological insecticide) against adult macadamia seed weevil under laboratory conditions. All isolates were pathogenic against adult weevils with M. anisopliae accession ECS1/BRIP 70272 and B. bassiana accession B27/BRIP 70267 causing 97.5% and 92.5% mortality 12 days after being treated at 1 × 107 conidia/mL. Isolates ECS1/BRIP 70272 and B27/BRIP 70267 had the shortest LT50 values of 5.13 days and 5.37 days respectively. The median lethal concentrations (LC50) for ECS1/BRIP 70272 and B27/BRIP 70267 were 1.48 × 105 and 1.65 × 105 conidia/mL respectively. Results of this study indicate that M. anisopliae accession ECS1/BRIP 70272 and B. bassiana accession B27/BRIP 70267 have considerable potential for K. macadamiae control, and should be developed into biological insecticides for integration into macadamia pest management programs.

Source Attribution of Salmonella in Macadamia Nuts to Animal and Environmental Reservoirs in Queensland, Australia.

Salmonella enterica is a common contaminant of macadamia nut kernels in the subtropical state of Queensland (QLD), Australia. We hypothesized that nonhuman sources in the plantation environment contaminate macadamia nuts. We applied a modified Hald source attribution model to attribute Salmonella serovars and phage types detected on macadamia nuts from 1998 to 2017 to specific animal and environmental sources. Potential sources were represented by Salmonella types isolated from avian, companion animal, biosolids-soil-compost, equine, porcine, poultry, reptile, ruminant, and wildlife samples by the QLD Health reference laboratory. Two attribution models were applied: model 1 merged data across 1998-2017, whereas model 2 pooled data into 5-year time intervals. Model 1 attributed 47% (credible interval, CrI: 33.6-60.8) of all Salmonella detections on macadamia nuts to biosolids-soil-compost. Wildlife and companion animals were found to be the second and third most important contamination sources, respectively. Results from model 2 showed that the importance of the different sources varied between the different time periods; for example, Salmonella contamination from biosolids-soil-compost varied from 4.4% (CrI: 0.2-11.7) in 1998-2002 to 19.3% (CrI: 4.6-39.4) in 2003-2007, and the proportion attributed to poultry varied from 4.8% (CrI: 1-11) in 2008-2012 to 24% (CrI: 11.3-40.7) in 2013-2017. Findings suggest that macadamia nuts were contaminated by direct transmission from animals with access to the plantations (e.g., wildlife and companion animals) or from indirect transmission from animal reservoirs through biosolids-soil-compost. The findings from this study can be used to guide environmental and wildlife sampling and analysis to further investigate routes of Salmonella contamination of macadamia nuts and propose control options to reduce potential risk of human salmonellosis.

Sensory properties of yellow pea and macadamia honeys from conventional and flow hive extraction methods.

BACKGROUND: The process of harvesting honey is time consuming and labor intensive. A new system, the Flow Frame, has drastically simplified the harvesting process, enabling honey to be extracted directly from the hive with minimal processing. The sensory profile of honey is influenced, first, by botanical origin and subsequently by processing and storage parameters. A reduction in harvest processing may thus influence the sensory profile of honey harvested from FFs compared to that of honey produced from conventional processing. To test this hypothesis, two monofloral honeys (macadamia and yellow pea) were harvested from FFs, or by conventional honey extraction. Sensory profiling using conventional descriptive analysis was carried out for each floral source with an experienced trained panel. RESULTS: The two monofloral honeys harvested using the FF system had significantly (p < 0.05) higher floral and cleaner aftertaste sensory scores than the honey extracted using commercial (C) methods that involve the use of heat and centrifugation. CONCLUSION: The flow system retains honey's natural sensory properties compared to harvesting methods that require heat and centrifugation. © 2019 Society of Chemical Industry.

Improvement in nutritional quality and thermal stability of palm olein blended with macadamia oil for deep-fat frying application.

This study aimed to investigate the suitability of refined bleached deodorized palm olein oil (RBD POO) and Macadamia integrifolia oil (MO) blend as deep-fat frying substitute. Oxidative and hydrolytic stability of MO, POO and blends during 15 days of storage under accelerated oxidation condition (65 °C ± 1) studied by assessing free fatty acids (FFA), peroxide (PV), anisidine (AV) and TOTOX (TV) values. Blends formulated with POO: MO at 100:0, 75:25, 50:50, 25:75 and 0:100. Blending significantly affected the fatty acid profile, smoke point, FFA, PV, AV and TV of all samples (p < 0.05). Both independent variables (storage time and type of oil) and their interaction had significant effect on FFA, PV, AV and TV (p < 0.05). On day 15, the highest and the lowest FFA observed for MO and POO, respectively. The significant difference between FFA of MO and POO before incubation and day 15th was due to role of refining process in elimination of initial FFAs from POO. Results of PV, AV and TV showed that the highest and least changes were presented by POO and MO, respectively. Oil blends containing higher proportions of MO with a great percentage of monounsaturated and less polyunsaturated fats were more stable against oxidation. Also, presence of antioxidants played significant role against MO oxidation. From this research, blending POO with MO improved induction period of the blends and inhibited primary and secondary oxidation products formation. Blend 25:75 met the qualitative and nutritional criteria and suggested for formulation of a functional oxidative stable frying medium.

Biomass-derived carbon composites for enrichment of dilute methane from underground coal mines.

Ventilation air methane (VAM), which is the main source of greenhouse gas emissions from coal mines, has been a great challenge to deal with due to its huge flow rates and dilute methane levels (typically 0.3-1.0 vol%) with almost 100% humidity. As part of our continuous endeavor to further improve the methane adsorption capacity of carbon composites, this paper presents new carbon composites derived from macadamia nut shells (MNSs) and incorporated with carbon nanotubes (CNTs). These new carbon composites were fabricated in a honeycomb monolithic structure to tolerate dusty environment and to minimize pressure drop. This paper demonstrates the importance of biomass particle size distributions when formed in a composite and methane adsorption capacities at low pressures relevant to VAM levels. The selectivity of methane over nitrogen was about 10.4 at each relevant partial pressure, which was much greater than that (6.5) obtained conventionally (at very low pressures), suggesting that capturing methane in the presence of pre-adsorbed nitrogen would be a practical option. The equilibrium and dynamic performance of biomass-derived carbon composites were enhanced by 30 and 84%, respectively, compared to those of our previous carbon fiber composites. In addition, the presence of moisture in ventilation air resulted in a negligible effect on the dynamic VAM capture performance of the carbon composites, suggesting that our carbon composites have a great potential for site applications at coal mines because the cost and performance of solid adsorbents are critical factors to consider.

BSACI guideline for the diagnosis and management of peanut and tree nut allergy.

Peanut nut and tree nut allergy are characterised by IgE mediated reactions to nut proteins. Nut allergy is a global disease. Limited epidemiological data suggest varying prevalence in different geographical areas. Primary nut allergy affects over 2% of children and 0.5% of adults in the UK. Infants with severe eczema and/or egg allergy have a higher risk of peanut allergy. Primary nut allergy presents most commonly in the first five years of life, often after the first known ingestion with typical rapid onset IgE-mediated symptoms. The clinical diagnosis of primary nut allergy can be made by the combination of a typical clinical presentation and evidence of nut specifc IgE shown by a positive skin prick test (SPT) or specific IgE (sIgE) test. Pollen food syndrome is a distinct disorder, usually mild, with oral/pharyngeal symptoms, in the context of hay fever or pollen sensitisation, which can be triggered by nuts. It can usually be distinguish clinically from primary nut allergy. The magnitude of a SPT or sIgE relates to the probability of clinical allergy, but does not relate to clinical severity. SPT of ≥ 8 mm or sIgE ≥ 15 KU/L to peanut is highly predictive of clinical allergy. Cut off values are not available for tree nuts. Test results must be interpreted in the context of the clinical history. Diagnostic food challenges are usually not necessary but may be used to confirm or refute a conflicting history and test result. As nut allergy is likely to be a long-lived disease, nut avoidance advice is the cornerstone of management. Patients should be provided with a comprehensive management plan including avoidance advice, patient specific emergency medication and an emergency treatment plan and training in administration of emergency medication. Regular re-training is required.

Genome and transcriptome sequencing characterises the gene space of Macadamia integrifolia (Proteaceae).

BACKGROUND: The large Gondwanan plant family Proteaceae is an early-diverging eudicot lineage renowned for its morphological, taxonomic and ecological diversity. Macadamia is the most economically important Proteaceae crop and represents an ancient rainforest-restricted lineage. The family is a focus for studies of adaptive radiation due to remarkable species diversification in Mediterranean-climate biodiversity hotspots, and numerous evolutionary transitions between biomes. Despite a long history of research, comparative analyses in the Proteaceae and macadamia breeding programs are restricted by a paucity of genetic information. To address this, we sequenced the genome and transcriptome of the widely grown Macadamia integrifolia cultivar 741. RESULTS: Over 95 gigabases of DNA and RNA-seq sequence data were de novo assembled and annotated. The draft assembly has a total length of 518 Mb and spans approximately 79% of the estimated genome size. Following annotation, 35,337 protein-coding genes were predicted of which over 90% were expressed in at least one of the leaf, shoot or flower tissues examined. Gene family comparisons with five other eudicot species revealed 13,689 clusters containing macadamia genes and 1005 macadamia-specific clusters, and provides evidence for linage-specific expansion of gene families involved in pathogen recognition, plant defense and monoterpene synthesis. Cyanogenesis is an important defense strategy in the Proteaceae, and a detailed analysis of macadamia gene homologues potentially involved in cyanogenic glycoside biosynthesis revealed several highly expressed candidate genes. CONCLUSIONS: The gene space of macadamia provides a foundation for comparative genomics, gene discovery and the acceleration of molecular-assisted breeding. This study presents the first available genomic resources for the large basal eudicot family Proteaceae, access to most macadamia genes and opportunities to uncover the genetic basis of traits of importance for adaptation and crop improvement.