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.

Effects of Substrate Composition on the Growth Traits of Grafted Seedling in Macadamia (Macadamia integrifolia) Nuts.

Macadamia nut plantings in China are expanding year by year. In order to breed and promote superior varieties, this study analyzed the effects of different rootstocks and scions on the survival rate of grafted seedlings, and then selected the best substrate composition for plant growth. The results showed that the survival rate of the HAES788 variety as rootstock and Guire No. 1 as scion was the highest, reaching 96%. The optimal grafting time in December was better than that in March. Furthermore, among 16 substrate formulations, T12, T13, T15, and T16 had advantages of agglomerated soil and more well-developed root systems compared to the CK made of loess. The plant height, stem diameter, leaf length, leaf width, and dry weight of the aboveground and underground parts of the grafted seedlings planted in these substrate formulations were significantly higher than those plants planted in the CK. In addition, the substrate formulations T12, T13, T15, and T16 significantly improved the organic matter, total nitrogen, and total potassium content of the substrate soils, but little improvement was observed for total phosphorus content after 13 months. Overall, macadamia grafting times are best in December, with HAES788 and Guire No. 1 being the best rootstock and scion. The optimal substrate formulations are T12, T13, T15, and T16. This study provides a solid foundation for the production of high-quality macadamia plants.

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.

[Stability and organic carbon characteristics of soil aggregates under different ecosystems in karst canyon region].

Soil aggregates and their organic carbon distributions were studied under six ecosystems, i. e., farmland (short for ST), dry land (HD), grassland (CD), shrubbery (GC), plantation (RGL) and secondary forest (CSL), in a karst canyon region of China by a combination of field investigation and laboratory analysis. The result showed that, soil aggregates were dominated by particles with sizes>8 mm in the ecosystems except HD under dry sieving, and basically presented a trend of decreasing firstly, then increasing and finally decreasing along with particle sizes decreasing; while soil aggregates were dominated by particles with sizes > 5 mm in the ecosystems except HD under wet sieving and decreased with decreasing of particle sizes. The mean mass diameter (MMD) was in the order of ST>CD>RGL>CSL>GC>HD and the geometric mean diameter (GMD) was ST>CD>RGL>CSL>HD>GC by dry sieving, and MMD was RGL>CSL>GC>CD>ST>HD and GMD was CSL>RGL>GC>CD>ST>HD by wet sieving. Therefore, MMD and especially GMD of wet sieving were more accurate than that of dry sieving to evaluate soil aggregates quality in the karst cannon region. The fractal dimension (D) of mechanical stability in soil aggregates followed the order of CD>HD>ST>RGL>CSL>GC and the water stability was in the order of GC>CSL>RGL>HD> CD>ST. The higher the SOC content was, the larger values of D, MMD, GMD became, and the more sense the soil structure made. Soil organic carbon content was highest in the aggregate particles with sizes ranging from 0.25 to 0.053 mm, and the content in some particles with sizes > 5 mm was lowest. However, the contribution rate of particles with sizes > 5 mm was largest to soil organic carbon, which gradually decreased with the decrease of particle size.

[Characteristics of spatial variation of soil nutrients in sloping field in a gorge karst region, southwest China].

Based on a grid (20 m x 20 m) sampling, spatial heterogeneity and pattern of soil nutrients in sloping field in the gorge karst region, southwestern China, were explored by using classical statistics and geostatistics methods. The results showed that soil nutrient contents in slope field in the canyon karst region were more abundant, where pH value had a weak variation and the soil organic matter (SOM) had a moderate degree of variation. All the soil nutrients had moderate or strong variation with an order of available phosphorus (AP) > total potassium (TK) > SOM > alkaline nitrogen (AN) > total nitrogen (TN) > total phosphorus (TP) > available potassium (AK). All of the soil nutrients had a good spatial autocorrelation and the autocorrelation function performed in the same law of developing from positive to negative direction with the inflection point ranged from 80 to 100 m. In addition, the Moran's I was small for TK and AP while large for other nutrients. Characteristics of spatial variation differed among soil nutrients. Exponential model fitted best for TK and AP, in which the ratio of nugget to sill (C0/(C0 + C)) and the range (A) were small and the fractal dimension (D) was high, showed a strong spatial correlation. Spherical model fitted best for other soil nutrients, with C0/(C0 + C) , the range (A) and D showing a moderate autocorrelation. Kriging analysis clearly indicated that pH, SOM, TN, TP and AN were distributed in a concave pattern, while AP and AK had fragmented patch distribution. Therefore, vegetation, topography, human disturbance and strong heterogeneity of microhabitats are main factors leading to the differences in patterns of soil nutrients on the sloping land in the gorge karst region.

[Characteristics of soil microbial populations in depressions between karst hills under different land use patterns].

Based on the investigation and analysis of six soil microbial indices, eight soil conventional nutrient indices, six soil mineral nutrient indices, and 15 vegetation indices in the farmland, grassland, scrub, forest plantation, secondary forest, and primary forest in the depressions between karst hills, this paper analyzed the main soil microbial populations, soil microbial biomass carbon (MBC), nitrogen (MBN), and phosphorus (MBP) and their fractal characteristics, and the relationships of the soil microbes with vegetation, soil nutrients, and soil mineral components under different land use patterns. The soil microbial populations differed in their quantity and composition under different land use patterns. Primary forest and farmland had the highest quantity of soil microbial populations, while forest plantation had the lowest one. The three forests had a higher proportion of soil bacteria, the farmland, grassland, and scrub had a higher proportion of actinomycetes, and all the six land use patterns had a low proportion of soil fungi. Under the six land use patterns, the soil MBC, MBN, and MBP were all high, with the maximum in primary forest. There was a good fractal relationship between the soil MBC and microbial populations, but no fractal relationships between the soil MBN and MBP and the microbial populations. Significant relationships were observed between the soil microbes and the vegetation, soil nutrients, and soil mineral components, and the soil MBC had the closest relationships with the Shannon index of tree layer and the soil total nitrogen, Fe2O3, and CaO contents.