First report of Aloe root and stem rot caused by Phytophthora palmivora in Yunnan Province, China.

Aloe genus plants are perennial evergreen herb belonging to Liliaceae family which is widely used in food, medicine, beauty, and health care (Kumar et al. 2019). In August 2021, symptoms of root and stem rot was observed in approximately 20% of Aloe vera plantings in Yuanjiang County, Yunnan Province, China (23° 64' 53" N, 101° 99' 84" E). The most typical symptoms were stem and root rot, browning and necrosis of vascular tissues, gradual greening, and reddish-browning of leaves from bottom to top, abscission, and eventual plant death (Fig. S1). Therefore, to isolate and identify the pathogen, the plants showing the above symptoms were collected. The plant tissues were cut from the edges of root and stem lesions, followed by disinfection with 75% ethanol for 1 min, rinsed three times with sterilized distilled water, and cut into 3 × 3 mm small squares after excision of marginal tissues. The tissues were transferred to the oomycetes selective medium (Liu et al. 2022) and incubated at 28 °C in the dark for 3~5 days, and suspected colonies were purified. The colonies were then inoculated onto potato dextrose agar (PDA), V8-juice agar (V8), and oatmeal agar (OA) medium plates for morphological characteristics. Finally, 18 isolates with the same colonial and morphological characteristics were obtained from 30 lesioned tissue and one of them was named as ARP1. On PDA, V8 and OA medium plates, the ARP1 colonies were white. On PDA plate, the mycelia were dense and the colonies were petal-like; on V8 plate, the mycelia were cashmere and the colonies were radial or star-like. Whereas, on OA plate, the mycelia were cotton-like and the colonies were fluffy and radial (Fig. S2 A~C). Mycelium did not have septum with high branching and swelling. Sporangia were abundant, semi-papillate, varying in shape from ovoid-ellipsoid to long-ellipsoid, 18-26 × 45-63 µm (average: 22 × 54 µm, n = 30), sporangia released numerous zoospores from the papillate after maturation. The chlamydospores were spherical, 20-35 µm in diameter (average: 27.5 µm, n = 30) (Fig. S2 D~F). These morphological features were like those of the pathogenic species of the oomycetes (Chen et al. 2022). For the molecular characterization, the genomic DNA of the isolate was extracted using the cetyl trimethyl ammonium bromide method, and the translation elongation factor 1α (tef-1α) (Stielow et al. 2015), ß-tubulin (ß-tub) (Kroon et al. 2004) and internal transcribed spacer (ITS) (White et al. 1990) of isolated strain ARP1 were amplified using primer pairs EF1-1018F/EF1-1620R, TUBUF2/TUBUR1 and ITS1/ITS4, respectively. The tef-1α, ß-tub genes and ITS region of ARP1 were directly sequenced and their sequence information was deposited in GenBank under accession numbers OQ506129, OQ506127 and OQ449628. ARP1 was clustered on the same evolutionary branch with Phytophthora palmivora (Fig. S3). To confirm the pathogenicity of ARP1, the main root of A. vera was wounded to 1 cm long and 2 mm deep with a scalpel blade followed by inoculation with 50 ml suspension of ARP1 zoospores at a concentration of 1 × 106 spores / ml per potted plant, and an equal volume of water as control. All inoculated plants were placed in the greenhouse at 28°C, 12 h / 12 h light / dark. After 15 dpi, the inoculated plants showed typical symptoms of wilted and drooping leaves and stem and root rot, same as observed in the field condition (Fig. S4). After inoculation with ARP1, a strain with the same morphological and molecular characteristics as the original isolate was re-isolated, confirming Koch's postulates. To our knowledge, this is the first report of P. palmivora causing root and stem rot of A. vera in the study region. This disease could be a potential risk for aloe production and therefore appropriate management measures should be taken.

First report of apple root rot caused by Phytopythium vexans in China.

Apple production is of great economic importance in the fruit industry of China, where Yunnan Province is considered as a major producing area. A survey was conducted to identify apple trees that were problematic from March to November 2020 in Yunnan Province. Symptoms included smaller yellowing leaves, fewer sprouts per branch, browning and necrosis of the roots and lower parts of the stem bark, and wilting. 20% to 45% of apple trees were found infected and randomly scattered in the surveyed orchards. A total of 110 soil samples were collected from the root area of symptomatic apple trees in Tuanjie Town of Kunming City, Zhaotong City, and Malong District of Qujing City in Yunnan Province. Two grams of each soil sample was suspended in 400 ml of sterile water for three days and each soil extract was baited with two apple leaves (Red Fuji's). Following the baiting, those leaves were cut into 10 pieces (5mm×5mm), surface-sterilized with 70% ethanol for 30 seconds, rinsed three times with sterile water, and then air-dried. Each leaf piece was placed in a Petri dish with the oatmeal agar medium containing PCNB 20 mg/ml, rifampicin 20 mg/ml, and then incubated at 25℃ in the dark for 3 days. A mycelial agar plug was picked from the edge of the colonies and transferred to a fresh Potato Dexrose Agar (PDA) plate. Seventy colonies with similar growing characteristics were isolated from the 110 soil samples. Three isolates were retained for further analysis and named XLD8-1, SD1, and YF2. After being cultivated on PDA plates and incubated at 25℃ in the dark for 4 days, their colonies were rose petal-type and white with dense aerial hyphae (Fig 1, A). In ten days of incubation, oogonium measuring 24.55 ± 1.9µm × 20.27 ± 2.3µm and sporangia measuring 21.65 ± 1.3µm × 19.35 ± 1µm were observed (Fig 1, C, D). The total DNA of the isolates was extracted and amplified using three pairs of primers, ITS1/ITS4 (White et al. 1990), LROR/LR7 (LSU) (Vilgalys R, et al. 1990), and FM58/FM66 (COXⅡ) (Martin F N. 2000). The sequences were uploaded to GenBank (Accession No. OL960234, OK037658, OK052604 for ITS, OL960388, OM838413, OM838314 for LSU, and OM962847, OM962848, OM962849 for COXⅡ). ITS sequences of the three isolates (XLD8-1, SD1, YF2) showed 99.87%,99.87%, 99.87% similar to Pp. vexans (Accession No. AB468784, AB468784, and AM701801). LSU sequences of the three isolates showed 99.92%, 99.72%, 100% similar to Pp. vexans (Accession No. EF426541, MT729990, and EF426541). COXⅡ sequences of the three isolates showed 100%, 99.81%, 99.81% similar to Pp. vexans (Accession No. GU133560). Based on the sequence similarity and morphology, the isolates were identified as Phytopythium vexans. Koch's postulates were conducted by wounding the bases of 3 apple seedlings (1-year-old Red Fuji's) with a cork borer. A plug of mycelium of the isolate XLD8-1 grown on PDA plates was placed on each wound (Fig 1, B). Controls were set up to use sterile agar plugs as an inoculum. Seedlings have incubated an incubator at 23-26°C under the alternating light and dark intervals, 12-hours of each. In 15 days, after were inoculated with XLD8-1 the roots and lower part of the stem bark of those seedlings became brownish and necrotic, and their epidermis was easily sloughed off (Fig 1, E-G). The pathogen isolated from the necrotic root tissues were identical to the isolate XLD8-1. Symptoms of apple growth decline caused by Pp. vexans were reported in Morocco (Jabiri Salma, et al. 2021). This experiment verified that Pp. vexans causes root rot of apple. In China, Fusarium sp. is usually considered the main pathogen causing apple root rot. However, the discovery of large numbers of apple trees that were infected by Pp. vexans in Yunnan Province and the confirmation of pathogenicity of Pp. vexans on apple seedlings have demonstrated for the first time that Pp. vexans could cause apple root rot as Fusarium spp does and become an incoming threat to the apple industry, which lays the foundation for study on the disease epidemiology and integrated management of apple root rot in China. References: Jabiri Salma, et al. 2021. Microorganisms, doi:10.3390/MICROORGANISMS9091916. Martin, F. N. 2000. Mycologia, 92(4), 711-727. Vilgalys R., et al. 1990. Journal of Bacteriology, 172:4238-4246 White, T. J., et al. 1990. PCR Protocols: a guide to methods and applications, 18: 315.

Acid-responsive properties of fibrils from heat-induced whey protein concentrate.

The heat-induced fibrils of whey protein concentrate (WPC) have demonstrated an acid-responsive property; that is, the fibrils went through formation-depolymerization-reformation as pH was adjusted to 1.8, 6.5, and back to 1.8. We investigated the microstructure, driving force, and thermal stability of 3.0% (wt) WPC nanofibrils adjusted between pH 6.5 and 1.8 twice. The results showed that the nanofibrils had acid-responsive properties and good thermal stability after reheating for 10h at 90°C and adjusting pH from 1.8 to 6.5 to 1.8. The content of WPC fibril aggregates was not much different with the prolongation of heating times during pH variation. Although the nanofibrils' structure could be destroyed only by changing the pH, the essence of this destruction might only form fiber fragments, polymers that would restore a fibrous structure upon returning to pH 1.8. A described model for the acid-responsive assembly of fibrils of WPC was proposed. The fibrils went through formation-depolymerization-reformation by weaker noncovalent interactions (surface hydrophobicity) as pH changed from 1.8 to 6.5 back to 1.8. However, the fibrils lost the acid-responsive properties because much more S-S (disulfide) formation occurred when the solution was adjusted to pH 6.5 and reheated. Meanwhile, fibrils still possessed acid-responsive properties when reheated at pH 1.8, and the content of fibrils slightly increased with a further reduction of α-helix structure.

Quality indices of the set-yoghurt prepared from bovine milk treated with horseradish peroxidase.

Horseradish peroxidase (HRP, EC 1.11.1.7) was applied to treat whole bovine milk in the presence or absence of ferulic acid (FA). The treated milk exhibited different rheological properties from the control milk, and was used to prepare set-yoghurt with commercial direct vat set starter. Some chemical, textural and rheological properties of the yoghurt prepared were measured and compared. Compared to that prepared with the control milk, the yoghurt prepared with the HRP- or HRP and FA-treated bovine milk exhibited an increased hardness and adhesiveness, lower syneresis extent, higher apparent viscosity, and higher storage modulus and viscous modulus. Observation of the microstructure of the yoghurt samples under scanning electron microscopy illustrated that HRP treatment of bovine milk led to the prepared yoghurt a more compact and uniform structure. The results in the present work stated that treatment of bovine milk with HRP in the presence of ferulic acid could be applied to improve the quality of set-yoghurt.

Optimization of some conditions of Neutrase-catalyzed plastein reaction to mediate ACE-inhibitory activity in vitro of casein hydrolysate prepared by Neutrase.

Casein hydrolysate was prepared by hydrolyzing casein with Neutrase and then modified by a Neutrase-catalyzed plastein reaction. The prepared hydrolysate had a degree of hydrolysis of 13.0% and exhibited ACE inhibition in vitro with an IC50 value of 40.4 µg⋅mL(-1). With the decreased amount of free amino groups of the modified hydrolysate as the response, some conditions of the plastein reaction including substrate concentration, enzyme to substrate ratio, reaction temperature and time were studied by single factor experiments and response surface methodology, and optimized finally as 62% (w/w), 3.0 kU⋅g(-1) peptides, 30 °C and 6.3 h, respectively. The maximum decreased amount of free amino groups of the modified hydrolysate prepared under these optimized conditions was 210.0 µmol⋅g(-1) peptides, while corresponding IC50 value was lowered to 14.7 µg⋅mL(-1). The present result indicates that Neutrase-catalyzed plastein reaction was capable of enhancing ACE-inhibitory activity in vitro of casein hydrolysate, and also highlights the importance of a forthcoming study to investigate the peptide compositions of the modified hydrolysate and the role of protease used in the plastein reaction.

Preparation and characterization of pea protein isolate-pullulan blend electrospun nanofiber films.

The objective of this work was to fabricate and characterize food-grade pea protein isolate (PPI) and carbohydrate polymer pullulan (PUL) nanofiber films by using green electrospinning technology. The effect of the blend ratios on the PPI/PUL solution properties (e.g. viscosity, surface tension and electrical conductivity) and morphology of the resulting electrospun nanofibers was investigated. The presence of PUL in the blends resulted in decreased apparent viscosity (P < 0.05), stable surface tension (42.09-46.26 mN/m) (P < 0.05) and lower conductivity of the solutions (P < 0.05), which were due to a better chain entanglement and decrease in the polyelectrolyte protein character, respectively, both factors were needed for uniform nanofiber (around 203 nm) formation. Rheological evaluation indicated a pseudoplastic behavior for all formulations. The Fourier transform infrared spectral changes and XRD patterns indicated that the protein and polysaccharide were well tangled in nanofibers. The results of the differential scanning calorimetry (DSC) indicate that thermal stability of the electrospun nanofiber films were improved in comparison to pure PUL. Finally, in order to expand the application range of the electrospun nanofiber films in future, thermal crosslinking method was conducted and water contact angles (WCAs) of the thermal treated nanofiber films exhibited better hydrophobic properties compared to the un-crosslinking samples.

Enhancement of the Textural and Gel Properties of Frankfurters by Adding Thermo-reversible or Thermo-irreversible Curdlan Gels.

The influence of different addition levels (0.1% to 0.5%) of thermo-reversible curdlan gels (TRC) and thermo-irreversible curdlan gels (TIRC) on the physicochemical and textural characteristics of frankfurters, as well as dynamic rheological properties of meat batters, was investigated. Increased percentages of TRC and TIRC were associated with lower cooking loss and quicker relaxation times, as well as superior emulsion stability, and higher L* -values and b* -values of frankfurters (P < 0.05). Moreover, with equal curdlan concentrations from 0.3% to 0.5%, TRC showed higher hardness and chewiness values than those with added TIRC (P < 0.05), but the gumminess, springiness, and resilience values were almost the same between each treatment (P > 0.05). The textural profile results were in strong agreement with the rheological data. Principal component analysis revealed that certain quality attributes were affected differently by the inclusion levels of TRC and TIRC. Additionally, the distinctive mechanism of the formation of the complex meat protein network by TRC or TIRC was also clarified and verified via scanning electron microscopy analysis. Further studies will investigate the molecular interactions of meat proteins with these two types of curdlan gels as a function of addition levels. PRACTICAL APPLICATION: Curdlan can form two different types of gels mainly depending on heating temperature, designated as thermo-reversible curdlan gels (TRC) and thermo-irreversible curdlan gels (TIRC), respectively. The addition of these two gels could improve the textural and gel properties of frankfurters, as well as improve the rheological profiles of meat batters. Each gel type invokes a different mechanism of influence on the formation of the complex meat protein network. Results indicate that TRC (mainly as an effective gelling agent) and TIRC (mainly as a potential fat-mimetic) can provide distinctive frankfurter formulations catered to the requirements preferred by different consumers.

Fast dissolving oral films for drug delivery prepared from chitosan/pullulan electrospinning nanofibers.

In this study, Chitosan/pullulan composite nanofiber fast dissolving oral films (FDOFs) were prepared via electrospinning technology. The ratio of chitosan/pullulan (C/P) had an influence on solution property and nanofiber morphology, with the increase of chitosan, viscosity and conductivity of solutions increased, the morphology obtained by scanning electron microscopy indicated that the diameter of nanofibers decreased initially then increased. The Fourier transform infrared spectra indicated hydrogen bond interactions between chitosan and pullulan molecules. X-ray diffraction analysis proved that electrospinning process decreased the crystallinity of materials. Thermal analysis showed that melting point, degradation temperature and glass transition temperature increased with the addition of chitosan content in the FDOF. Water solubility test proved that the FDOF can dissolve in water completely within 60 s. Finally, in order to prove its practicability in future, a model drug of aspirin was encapsulated in the FDOF successfully.