Patterns and drivers of heat production in the plant genus Amorphophallus.

Thermogenesis - the ability to generate metabolic heat - is much more common in animals than in plants, but it has been documented in several plant families, most prominently the Araceae. Metabolic heat is produced in floral organs during the flowering time (anthesis), with the hypothesised primary functions being to increase scent volatilisation for pollinator attraction, and/or to provide a heat reward for invertebrate pollinators. Despite in-depth studies on the thermogenesis of single species, no attempts have yet been made to examine plant thermogenesis across an entire clade. Here, we apply time-series clustering algorithms to 119 measurements of the full thermogenic patterns in inflorescences of 80 Amorphophallus species. We infer a new time-calibrated phylogeny of this genus and use phylogenetic comparative methods to investigate the evolutionary determinants of thermogenesis. We find striking phenotypic variation across the phylogeny, with heat production in multiple clades reaching up to 15°C, and in one case 21.7°C above ambient temperature. Our results show that the thermogenic capacity is phylogenetically conserved and is also associated with inflorescence thickness. Our study paves the way for further investigations of the eco-evolutionary benefits of thermogenesis in plants.

Functional validation of AaCaM3 response to high temperature stress in Amorphophallus albus.

Amorphophallus is a perennial monocotyledonous herbaceous plant native to the southwestern region of China, widely used in various fields such as food processing, biomedicine and chemical agriculture. However, Amorphophallus is a typical thermolabile plant, and the continuous high temperature in summer have seriously affected the growth, development and economic yield of Amorphophallus in recent years. Calmodulin (CaM), a Ca2+ sensor ubiquitous in eukaryotes, is the most important multifunctional receptor protein in plant cells, which affects plant stress resistance by participating in the activities of a variety of signaling molecules. In this study, the key gene AaCaM3 for the Ca2+-CaM regulatory pathway was obtained from A. albus, the sequence analysis confirmed that it is a typical calmodulin. The qRT-PCR results demonstrated that with the passage of heat treatment time, the expression of AaCaM3 was significantly upregulated in A. albus leaves. Subcellular localization analysis revealed that AaCaM3 localized on the cytoplasm and nucleus. Meanwhile, heterologous transformation experiments have shown that AaCaM3 can significantly improve the heat tolerance of Arabidopsis under heat stress. The promoter region of AaCaM3 was sequenced 1,338 bp by FPNI-PCR and GUS staining assay showed that the promoter of AaCaM3 was a high-temperature inducible promoter. Yeast one-hybrid analysis and Luciferase activity reporting system analysis showed that the AaCaM3 promoter may interact with AaHSFA1, AaHSFA2c, AaHSP70, AaDREB2a and AaDREB2b. In conclusion, this study provides new ideas for further improving the signal transduction network of high-temperature stress in Amorphophallus.

Unveiling the impact of nitrogen deficiency on alkaloid synthesis in konjac corms (Amorphophallus muelleri Blume).

BACKGROUND: Konjac corms are known for their alkaloid content, which possesses pharmacological properties. In the primary cultivation areas of konjac, nitrogen deficiency is a common problem that significantly influences alkaloid synthesis. The impact of nitrogen deficiency on the alkaloids in konjac corms remains unclear, further complicated by the transition from mother to daughter corms during their growth cycle. RESULTS: This study examined 21 alkaloids, including eight indole alkaloids, five isoquinoline alkaloids, and eight other types of alkaloids, along with the associated gene expressions throughout the development of Amorphophallus muelleri Blume under varying nitrogen levels. Nitrogen deficiency significantly reduced corm diameter and fresh weight and delayed the transformation process. Under low nitrogen conditions, the content of indole alkaloids and the expression of genes involved in their biosynthesis, such as tryptophan synthase (TRP) and tryptophan decarboxylase (TDC), exhibited a substantial increase in daughter corms, with fold changes of 61.99 and 19.31, respectively. Conversely, in the mother corm, TDC expression was markedly reduced, showing only 0.04 times the expression level observed under 10 N treatment. The patterns of isoquinoline alkaloid accumulation in corms subjected to nitrogen deficiency were notably distinct from those observed for indole alkaloids. The accumulation of isoquinoline alkaloids was significantly higher in mother corms, with expression levels of aspartate aminotransferase (GOT), chorismate mutase (CM), tyrosine aminotransferase (TAT), and pyruvate decarboxylase (PD) being 4.30, 2.89, 921.18, and 191.40 times greater, respectively. Conversely, in daughter corms, the expression levels of GOT and CM in the 0 N treatment were markedly lower (0.01 and 0.83, respectively) compared to the 10 N treatment. CONCLUSIONS: The study suggests that under nitrogen deficiency, daughter corms preferentially convert chorismate into tryptophan to synthesize indole alkaloids, while mother corms convert it into tyrosine, boosting the production of isoquinoline alkaloids. This research provides valuable insights into the mechanisms of alkaloid biosynthesis in A. muelleri and can aid in developing nitrogen fertilization strategies and in the extraction and utilization of alkaloids.

First Report of Nigrospora oryzae Causing Leaf Spot Disease on Amorphophallus albus in Shaanxi Province of China.

Amorphophallus albus P. Y. Liu & J. F. Chen is a typical cash crop widely planted in southwest China (Gao et al., 2022). In early August of 2021, a peculiar leaf spot disease was first detected on A. albus in Ankang Academy of Agricultural Sciences manufacturing base (32°69'N, 109°02'E), Shaanxi, China. Small irregular yellow-brown spots (1 to 2 mm) were observed on the surface of A. albus leaf. Following infection of the leaf, it expanded (3 to 5 mm) and became necrotic. Nine planting bases were investigated, and approximately 75% of plants were symptomatic during the rapid expansion period of bulb growth in Hanyin, Langao and Hanbin counties, Ankang City, Shaanxi, China. Higher disease incidence was observed at temperatures above 30℃ and humidity above 80%. Twenty-seven symptomatic tissues of infected leaves were first surface sterilized by immersion in 75% ethanol for 1 minute, followed by rinsing three times in sterile distilled water. The tissues were then cut into 4-5 mm pieces, plated on 1.5% potato dextrose agar (PDA), and incubated at 28±2°C. The hyphal tip from the growing edge of colonies cultured for three days at 28±2℃ was transferred to PDA to obtain pure cultures. Fungal colonies were white, then grey to black with an unevenly distributed, fast-growing aerial mycelium covering the petri dish within five days at 28±2℃. The colony turned dark brown when maintained in the dark at 28±2℃ after seven days, then grayish brown upon sporulation after 15 days (Fig.1f-g). Conidia were brown or black, smooth, spherical to sub-spherical, single-celled (8-12 µm × 10-13µm, average 9-11.5 µm in diameter, n=5µm). The nutritional hyphae exhibited septa, and a portion of the aerial hyphae formed a long, rough conidium, giving rise to a nearly spherical apical sac (Fig.1h). The surface gave rise to several small peduncles bearing clusters of surfaced spherical conidia (Fig.1i). Surfaced spherical conidia were generated on the surface of the small peduncle (Fig.1j). These morphological features were consistent with Nigrospora oryzae (Li et al., 2017). Genomic DNA was extracted from mycelia of the pathogen using an Ezup column fungal genomic DNA extraction kit (Sangon Biotech, Shanghai, China). To confirm the identity of the pathogen, the genomic fragments for the internal transcribed spacer (ITS), LSU (28S) and BenA gene of the isolate were amplified by PCR (Wang et al., 2017) and sent for sequencing. The resultant sequence (GeneBank ID of gene ITS, LSU, BenA are OR723825, OR775345, OR277316, respectively) were compared with the voucher specimens. BLAST results showed >99% identity with those of N.oryzae (GeneBank ID of N.oryzae strain LC2707 ITS, LSU, BenA are KX985954, KY806242, KY019481, respectively). A neighbor joining phylogenetic tree with the concatenated sequences of these genes showed that A-pb169 had the closest match with N. oryzae (Fig. 2). For pathogenicity testing, fifty plants in a period of rapid expansion of bulb growth were selected. Four leaves per plant were inoculated by sprayed till runoff with a conidial suspension of the pathogen (50 µL, 1×106 conidia/ml sterile water), and incubated at 30±2℃ and 80 ± 5% humidity. Control plants received sterile water. On the third day after inoculation, a yellow-brown spot appeared on leave surfaces, the spot gradually expanded; the infection rate was 90 to 95%. Fifteen days after inoculation, infected leaves showed symptoms like those observed in the field, whereas 100 control leaves sprayed with sterile water remained symptomless (Fig.1 a-e). The pathogen was reisolated from infected leaves and confirmed as N. oryzae by morphology and molecular identification. To our knowledge, this is the first report of leaf spot disease of A. albus caused by N. oryzae in China. Since its one of the major cash crops of the southeastern China, further work is necessary to determine its spread and economic impact as well as developing sustainable disease management options.

First Report of Paramyrothecium breviseta Causing Target Spot on Amorphophallus muelleri in Yunnan, China.

Amorphophallus muelleri is an Araceae plant with perennial tuber, widely used in food, pharmaceutical and chemical industry due to its richness in glucomannan. In April 2022, an outbreak of a target spot on A. muelleri plantlets was observed in a nursery in Ruili, Yunnan, China. The leafstalks of the diseased plantlets in the nursery turned brown and decayed (Fig.1 A-B), then gradually some water-soaked spots on the true leaves developed along the veins (Fig.1 A). Subquencely, the spots on the true leaves turned dark green to white-grayish in the center, which formed light to dark brown concentric rings with a target-like appearance surrounded by a yellow halo (Fig.1 C). When the temperature was 20-34℃ and the relatively humidity was 25-80%, dark-green to black sporodochia with white hypha appeared on the lower and upper leaf surfaces. Finally, 5-8% of the plants surveyed on 800 m2 of one-year-old plantlets in the nursery showed the symptoms and some plants with infected leafstalks would be death. Similar symptoms were also observed on about 10% of the transplanted plants surveyed on 12000 m2 (1.2 ha) of two-year-old plantlets in the field. Five diseased leaves from five distinct plantlets in the nursery were collected for pathogen isolation. Leaf pieces(5 x 5 mm) were cut from the edge of necrotic lesions, and surface-sterilized with 2.5% sodium hypochlorite for 1 min, 75% ethanol for 30 s, then rinsed 5 times by sterilized distilled water, finally put the leaf pieces on sterilized filter paper for 3-5 minutes to dry them and transferred onto potato dextrose agar (PDA) in petri dishes at 25℃ for three days. Five pure cultures identical to colony and conidial characteristics were isolated from five individual plants. The representative pure culture (M1) was grayish-white and circular colonies were 7.50 cm in diamter after 15 days at 25℃, with dark green concentric rings of sporodochia, the dorsal view of the colonies were yellowish. Conidia were aseptate, smooth, cylindrical, 5.00-6.25 (5.71) x 1.25-1.67 (1.63) µm (n = 20) rounded at both ends. A spore suspension (1 x 106 spores/ml) was prepared by harvesting spores from 15-day-old cultures grown in the dark at 25℃, then a thirty-ml of spore suspension was sprayed on the healthy leaves of 10 two-year-old plantlets. Thirty-ml of sterile water was sprayed on the healthy leaves of another 10 seedlings and used as the control. All seedlings were placed in a nursery at 20 to 34℃ and a relative humidity of 25 to 80%. Similar symptoms (Fig.1 D-F) to those observed in the nursery and field developed on all the 10 seedlings inoculated with M1 after two days, but not on the control leaves. The pathogenicity tests were repeated for three times. Fungal cultures reisolated from the infected leaves were identical to the original colonies and conidia, completing Koch's postulates. The internal transcribed spacer (ITS, primers ITS1 and ITS4) region of ribosomal DNA (OQ553785), calmodulin (cmdA, primers CAL-228F and CAL2Rd)(OQ559103), RNA polymerase II second largest subunit (rpb2, primers RPB2-5F2 and RPB2-7cR) (OQ559104) and ß-tubulin (tub2, primers Bt2a and Bt2b) (OQ559105) of M1 had 100%, 98.52%, 98.98% and 98.98% identity with the sequences of Paramyrothecium breviseta CBS544.75 (KU846289 for ITS, KU846262 for cmdA, KU846351 for rpb2, and KU846406 for tub2), respectively. In the phylogenic tree based on ITS, cmdA, rpb2 and tub2 gene sequences, the pure culture M1 clustered with P. breviseta CBS544.75, SDBR-CMU387, DRL4 and DRL3, which has been reported as the pathogen of leaf spot of Coffea arabica in China, C. canephora in China and Thailand (Wu et al. 2021; Withee et al. 2022). Molecular and morphological observations showed the pure culture M1 were P. breviseta (Withee et al. 2022), in addition the disease was named as target spot dueing to the typical target symptom on the leaves. To our knowledge, this is the first report of P. breviseta on A. muelleri from Yunnan, China, as well as worldwide. This disease can caused serious economic losses of A. muelleri dueing to that it can result 5-8% death of the plants in the nursery.

First Report of Cercospora chevalieri Causing A Leaf Spot Disease on Amorphophallus konjac in China.

Amorphophallus konjac, commonly called voodoo lily, is a cash crop widely cultivated in southwest China (Gao et al. 2022). In August 2022, leaf spot symptoms were observed in a field (1 ha) located at Fuyuan (25.67°N; 104.25°E), Yunnan, China, resulting in substantial economic losses. Brown lesions, with an incidence ranging from 20 to 40%, typically had a whitish or gray center and were surrounded by yellow halos. Microscopic observations of the spots revealed anamorphic species Cercospora chevalieri. Conidiophores were 50-150 × 4-7 µm, cylindrical, unbranched, smooth-walled, pale brown and aggregated in dense fascicles arising from a brown stroma. The conidiogenous cells were integrated, terminal or intercalary, pale brown to brown and proliferated sympodially. The conidiogenous loci were thickened and darkened, and 2-3 µm in diam. The conidia were formed singly, obclavate-cylindrical, 90-160 × 5-7 µm, with an average of 130 × 6 µm (n = 30), 6-11 septa, thin-walled, smooth, hyaline or subhyaline, straight or curved with an obtuse apex and obconically truncate base, with thickened and darkened hilum. These morphological characteristics matched those of C. chevalieri, the causal agent of leaf spot on A. paeoniifolius (Braun et al. 2014; Saccardo et al. 1913). A conidial suspension in sterile water from lesions was used to inoculate water agar, and germinated conidia were transferred to potato dextrose agar（PDA) and incubated at 27°C for 7 days. Induction of sporulation was unsuccessful using PDA, as well as malt extract agar, potato sucrose agar and synthetic nutrient-poor agar. Two out of ten isolates were selected for molecular identification and pathogenicity assay. Genomic DNA from two pure isolates (KUNCC22-12536 and KUNCC22-12537) was extracted for PCR and amplified with primers for the internal transcribed spacers (ITS: ITS1/ITS4), calmodulin (CMD: CAL228F/CAL2Rd), translation elongation factor 1-alpha (TEF1-α: 728F/986R), actin (ACT: 512F/783R), histone H3 (HIS3: CYLH3F/CYLH3R), beta-tubulin gene (TUB2: BT-1F/BT-1R) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH: Gpd1/Gpd2), respectively (Vaghefi et al. 2021). The newly generated sequences for ITS (OP719153/OP719154), CMD(OP740904/OP740905), TEF1-α (OP740910/OP740911), ACT (OP740902/OP740903), HIS3 (OP740908/OP740909), TUB2 (OP740912/OP740913), GAPDH (OP740906/OP740907) of C. chevalieri were submitted to GenBank. So far, no sequence data of C. chevalieri were available in the GenBank database. As expected, most genes (TEF1-α, ACT, CMD, HIS, TUB2 and GAPDH) showed 91 to 95% identity to their best hits within species of the genus Cercospora. The phylogenetic tree showed that sequences retrieved from two isolates obtained from the A. konjac leaf spots clustered together within Cercospora forming a strongly supported clade. To test Koch's postulates, ten four-month-old healthy A. konjac plants grown in pots were used for a pathogenicity test in a greenhouse. One leaf of each plant was inoculated with mycelial plugs, and one leaf was inoculated with a sterile PDA plug. These plants were enclosed in plastic bags for 72 h. Only leaves inoculated with mycelium plugs produced brown lesions, which appeared after 10 to 14 days on inoculated leaves. Control plants treated with sterile PDA plugs remained asymptomatic. This experiment was repeated twice with the same results. C. chevalieri was reisolated from infected leaves and identified based on morphology and Sanger sequencing of the ITS region. To our knowledge, this is the first report of C. chevalieri causing leaf spot on A. konjac and the first report of this species from China (Braun et al. 2014), which provides key information for diagnosis and management of this disease.

Characterization and shelf life of synbiotic drink powder from porang (Amorphophallus muelleri).

Amorphophallus muelleri BI was included in the Araceae family, which is a type of tuber. It is a tuber with high potential due to its abundant bioactive compounds. Amorphophallus muelleri BI flour (AF) contains a high glucomannan and carbon compounds that serve as nutrients for probiotic bacteria. Although Amorphophallus muelleri BI thrives in Indonesia, its utilization rate in the country remains relatively low and haven't been any studies conducted regarding synbiotic powder from AF. The primary objective of this research is to develop a synergistic beverage enriched with varying concentrations of Amorphophallus muelleri BI as a prebiotic and LA as probiotic (synbiotic). The process starts with culture preparation, synbiotic drink process, synbiotic and microencapsulation, includes the examination of solubility, proximate analysis, calorie content, viability, and shelf life. Results showed that the proximate and solubility had no significant effect. Synbiotic drink powder from AF can be produced using spray dry technology. The highest LA growth was observed when augmenting the AF quantity at a 0.4% concentration, which can be seen from the viability parameter with a value of 7.29 log CFU/g. Samples shelf life at -21 and 3 °C with LA viability critical parameter was determined to be 4 days.

Bacterial community structure in the rhizosphere of fungi-infected Amorphophallus titanum.

The rhizosphere is a narrow soil area directly affected by plant root exudates. Microbes inhabiting the rhizosphere have been widely studied for their beneficial effects on plant nutrition, growth, and disease prevention. Many factors affect the rhizosphere microbial composition, including plant pathogen infection. Here, we analyzed the bacterial community structure in the rhizosphere of fungi-infected Amorphophallus titanum. Soil samples were collected from rhizosphere and non-rhizosphere areas of fungi-infected A. titanum. The 16S metagenomic analysis was conducted to investigate the bacterial community of the samples by amplifying the V3-V4 region. The results showed that the phylum Firmicutes was prevalent in the rhizosphere, whereas the phyla Proteobacteria, Acidobacteria, and Actinobacteria were limited. Some major fungal genera were isolated from infected tubers and rhizosphere soil of A. titanum, including Trichoderma sp., Aspergillus sp., Perenniporia sp., and Cerrena sp. The fungal-isolate Aspergillus spp. is a well-known agricultural pest in several reports. While Cerrena sp. was reported to be pathogenic in plants, including the family of Arecaceae. Overall, the data revealed a potential relationship between fungal infections and the dominant bacterial community in the rhizosphere of A. titanum. Additionally, this research may contribute to the development of microbe-based technology to mitigate diseases in A. titanum.

First report of Fusarium tricinctum Causing Leaf Blight on Amorphophallus konjac in Hunan Province of China.

Konjac (Amorphophallus konjac) is a perennial herbaceous plant of the Araceae family, cultivated mainly in south-western China and used extensively for weight loss (Chua et al. 2010). In June 2022, leaf blight was detected on a 2,00 ha A. konjac plantation in Chenxi County, Huaihua City, Hunan Province. It infected almost 20% of the area under cultivation and tends to occur each year during warm, humid weather from May to July, causing significant economic losses to A. konjac production. There were small brown spots on the leaves which gradually spread to form irregular brown lesions. In severe cases the entire plant turned yellow and died. Nine samples were collected randomly from different plants in three plantation forests to isolate the pathogens. They were washed with sterile water and the lesions were excised. They were subsequently disinfected with 3% hydrogen peroxide for 30 s, 75% ethanol for 90 s and rinsed three times with sterile water. The cut sections were then placed on water agar plates and grown in the dark in a constant temperature incubator at 28℃ for 3-5 days, when mycelia grew they were transferred to potato dextrose agar medium and grown in the dark at 28℃ for 3-5 days. Eleven purified fungal isolates were obtained, ten of which looked like Fusarium (90.9% isolation rate), and three representative isolates (MY5, MY7 and MY9) were chosen for further study. The fungal colonies initially appeared white and gradually turnned dark red. Macroconidia were crescent-shaped, elongated, slightly curved and had 2 to 4 septations, with a predominance of 3 septations. They measured 15.540 to 42.083 × 2.760 to 4.558 µm (n=100). Microconidia were oval or pyriform, with a maximum of one septum and measured 6.135 to 24.990 × 2.158 to 4.412 µm (n=100). Two genetic regions, the translation elongation factor-1 (TEF1-α) and RNA polymerase II largest subunit (RPB1) genes, were amplified and sequenced to verify the identity of the fungus (Qiu et al. 2023). The universal primers TEF1-F/R, G2R/Fa were used for amplification and sequencing, and the sequences were submitted to GenBank (TEF1-α: OR545395, OR545397, OR545399; RPB1: OR545394, OR545396, OR545398). A joint phylogenetic tree of the two genes was constructed and analysis showed that the three isolates were significantly clustered with Fusarium tricinctum. Based on the results of morphological identification and phylogenetic tree analysis, the three isolates were identified as F. tricinctum. Pathogenicity tests were carried out on 12 uniformly growing leaf expansion stages of konjac plants，each inoculated with five young leaves. Mycelial blocks of 6 × 6 mm grown on PDA media for 5 days were placed on the surface of the leaves, while sterile PDA blocks were placed on the control plant. After 10 days of rearing the treated plants in a constant temperature chamber at 28°C and 90% relative humidity, the lesions appeared and the pathogens re-isolated from the diseased tissues had the same morphological characteristics as representative isolates. F. tricinctum has been shown to be the major pathogenic fungus causing leaf blight in wheat (Triticum aestivum L.) (Castañares et al. 2011) and orchardgrass (Dactylis glomerata L.) (Wu et al. 2020). To our knowledge, this is the first time in the world that F. tricinctum has been reported to cause leaf blight in A. konjac. This research could provide a foundation for future control of leaf blight disease.

First Report of Anthracnose Caused by Colletotrichum camelliae on Amorphophallus konjac in Hunan Province of China.

Amorphophallus konjac is a crop in the family Araceae, which is widely cultivated in Hunan, Yunnan, and Guizhou in China. A. konjac flour is highly valuable economically as a product for weight reduction. In June 2022, a new leaf disease was discovered in an understory A. konjac plantation in Xupu County, Hunan Province, China, where 2,000 hm2 of A. konjac had been planted. Approximately 40% of the total cultivated area exhibited symptoms. The disease outbreaks occurred during warm and wet weather (May to June). In the early stages of infection, small brown spots appeared on the leaves and then gradually spread into irregular lesions. There was a light yellow halo around the brown lesions. In severe cases, the whole plant gradually turned yellow and died. Six symptomatic leaf samples were collected from three different fields in Xupu County to isolate the causal agent. They were rinsed with sterile water, and the lesions were cut off. The lesions were rinsed in 3% hydrogen peroxide for 30 s followed by a 90 s treatment in 75% alcohol. They were then rinsed five times in sterile water, placed on water agar plates and incubated for 2-3 days at 28°C. After the mycelium had grown, they were transferred to potato dextrose agar (PDA) plates and incubated for 3-5 days at 28°C. In total, ten isolates were obtained, and seven of them were Colletotrichum (isolation frequency 70%). Three representative isolates (HY1, HY2, and HY3) were selected for further study. This fungus grew as circular white colonies, which then became grey. The older colonies looked like cotton and had dense aerial hyphae. The conidia were cylindrical, lacked a septum, and were thin-walled. They measured 14.04 to 21.58 × 5.89 to 10.40 µm (n=100). To further confirm its fungal identity, the fungus was amplified and sequenced using six genetic regions, including ß-tubulin (TUB2), actin (ACT), the internal transcribed spacer (ITS), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), calmodulin (CAL) and chitin synthase (CHS). The universal primers BT2a/TUB2R, ACT512F/ACT783R, ITS4/ITS5, GDF/GDR, CL1C/CL2C, CHS79F/CHS345R were used for amplification (Weir et al. 2012), sequenced by the Sanger chain termination method, and submitted to GenBank (TUB2: OQ506549, OQ506544, OP604480; ACT: OQ506551, OQ506546, OP604482; ITS: OQ457036, OQ457498, OP458555; GAPDH: OQ506553, OQ506548, OP604484; CAL: OQ506552, OQ506547, OP604483; CHS: OQ506550, OQ506545, OP604481). An analysis of a joint phylogenetic tree that was constructed using the six genes showed that the three isolates clearly clustered with Colletotrichum camelliae (syn. Glomerella cingulata f. sp. camelliae) strain ICMP 10646 (GenBank: JX010437.1, JX009563.1, JX010225.1, JX009993.1, JX009629.1, JX009892.1) and HUN1A4 (GenBank: KU252173.1, KU251646.1, KU251565.1, KU252019.1, KU251838.1, KU251913.1). HY3 was used as a representative strain for the pathogenicity test on the leaves of A. konjac from the whole plant. PDA blocks that were 6 × 6 mm and had been cultured for 5 d were placed on the leaf surface, and sterile PDA blocks were used as the control group. The climate chamber was maintained at 28°C at all times, and 90% relative humidity was maintained. The pathogenic lesions appeared after 10 days of inoculation. The pathogen that was re-isolated from the diseased tissues had the same morphological characteristics as HY3. Thus, Koch's postulates were fulfilled. C. camelliae has been shown to be the primary pathogenic fungus responsible for anthracnose in tea (Ca. sinensis (L.) O. Kuntze) (Wang et al. 2016) and camellia oleifera (Ca. oleifera Abel.) (Li et al. 2016). Anthracnose caused by Colletotrichum gloeosporioides has been reported on A. konjac (Li. 2021). However, to our knowledge, this is the first report in China and worldwide that C. camelliae causes anthracnose on A. konjac. This research lays the foundation for future research to control this disease.

First Report of Achromobacter Xylosoxidans Causing Stem Soft Rot on Amorphophallus Konjac in China.

Amorphophallus konjac is one of the important commercial crops cultivated in south China and has long been used as a food source and a traditional medicine, because it is the only species with glucomannan and other trace elements (Ban et al. 2009; Melinda et al. 2010). In June of 2021, an outbreak of stem soft rot disease was observed on A. konjac plants in more than 2,000 square meters of agricultural planting fields in the Fuyuan country (25°34'50″N, 104°04'21″E), Qujing City, Yunnan Province, China. The disease incidence ranged from 30 to 35% in severely infested fields. The diseased plants displayed the first symptoms were damp brown spots. As the brown spots expanded, dark brown water stains appeared at the basal part of the stem and the bulbs were rotting with a foul smell, gradually extending to the underground parts. Progressively, the whole plants wilted and collapsed, and even the plants ultimately died. To identify the pathogen, symptomatic stems were cut into pieces, surface sterilized with 75% (v/v) ethanol, and placed on LB (tryptone/yeast extract/NaCl) medium for 24 to 48 hours at 28 ± 2°C. Six single-colony isolates were obtained from the diseased stems. The colonies on LB present a raised milky white opaque colonies moisture on the surface, round and convex in shape, with neat edges. Scanning electron microscopy showed that the cells were short rods (0.3∼0.5) × (1.9∼2.1)µm in size without any flagellum and were often arranged in pairs or clusters at certain angles. The 16S rDNA sequence of the randomly selected strain MY-G1 with primers 27F/1492R (Ying et al. 2012) and the housekeeping genes nusA, eno, lepA and nuoL (Spilker et al. 2012) were amplified and sequenced. The 16S rDNA sequence of the 1326 bp product was deposited in GenBank (accession no. ON786717) and showed a 99.77% similarity to A. xylosoxidans strain E2 (accession no. MK849863.1). The nusA (OP680477), eno (OP680479), lepA (OP680481) and nuoL (OP680482) sequences showed 94.71%, 97.24%, 94.64% and 95.95% similarity to A. xylosoxidans strain DN002 (accession no. CP045222.1), respectively. The phylogenetic trees built based on 16S rRNA and the nusA-eno-lepA-nuoL multilocus analysis showed the isolate MY-G1 to cluster with A. xylosoxidans. Based on morphological and molecular analysis, the isolated MY-G1 was identified as A. xylosoxidans, which indicates that MY-G1 is a new strain of A. xylosoxidans. Pathogenicity tests were confirmed on the stem and petiole of one-year-old A. konjac. The wounds were made by puncturing with a MY-G1 bacteria suspension containing 108 CFU/ml (15ul/inoculation site). As a negative control, control seedlings were injected with the same amount of sterilized distilled water. Control and inoculated seedlings (each six) were kept in greenhouses and watered as needed in controlled conditions: 28°C, 75% relative humidity. Inoculated seedlings presented similar symptoms of stem soft rot, inner medulla disintegration, and wilt of leaves on developed within 3 to 9 days. The bacterial pathogen was re-isolated from inoculated seedlings and identified by morphological and molecular methods to fulfill Koch's postulates test. According to previous research, A. xylosoxidans can cross-kingdom infect animals and plants (Aisenberg et al.,2004; Ye et al.,2018). To the best of our knowledge, this is the first report of A. xylosoxidans causing stem soft rot of A. konjac in China, expanding the known pathogen for the soft rot of A. konjac, and also the host range of A. xylosoxidans.

The Effect of κ-Carrageenan Proportion and Hot Water Extract of the Pluchea indica Less Leaf Tea on the Quality and Sensory Properties of Stink Lily (Amorphophallus muelleri) Wet Noodles.

The study aims to determine the effect of the proportion of κ-carrageenan and the hot water extract of pluchea leaf tea on the quality and sensory properties of stink lily wet noodles. The research design is a randomized block design with two factors, i.e., the difference in the proportion of κ-carrageenan (K) (0, 1, 2, and 3% w/w) and the addition of the hot water extract of the Pluchea indica Less leaf tea (L) (0, 15, and 30% w/v), with 12 treatment levels (K0L0, K0L1, K0L2, K1L0, K1L1, K1L2, K2L0, K2L1, K2L2, K3L0, K3L1, K3L2). The data are analyzed by the ANOVA at p < 5% and continued with the Duncan's multiple range test at p < 5%, and the best treatment was determined by the spider web method based on sensory assay by a hedonic method. The proportions of κ-carrageenan and the concentration of pluchea tea extract had a significant effect on the cooking quality and sensory properties. However, the interaction of the two factors affected the swelling index, yellowness (b*), chroma (C), hue (h), total phenol content (TPC), total flavonoid content (TFC), and DPPH free radical scavenging assay (DPPH). The best treatment of wet noodles was K2L0, with a preference score of 15.8. The binding of κ-carrageenan and phenolic compounds to make a networking structure by intra- and inter-disulfide bind between glucomannan and gluten was thought to affect the cooking quality, sensory properties, bioactive compounds (TPC and TFC), and DPPH.

Chemical Profiling and Evaluation of Antioxidant and Anticancer Potential of Tuber Crop Amorphophallus commutatus var. wayanadensis.

Cancer and cancer-related diseases are a global health concern in the present scenario. Functional food and nutraceuticals are considered as a boon towards cancer management. Amorphophallus commutatus var. wayanadensis (ACW) is an herbaceous plant used by the local communities of Wayanad, India, for food and primary healthcare. Various radical scavenging and reducing power assays were undertaken to evaluate the antioxidant activity of methanolic extract of ACW (MEAC). In vitro anticancer activity was evaluated against HT-29 cell line by MTT assay, morphological analysis, DNA fragmentation assay and cell cycle analysis. Caspase and COX-2 enzyme assays were conducted to examine the underlying mechanism. Studies on Ehrlich Ascites Carcinoma (EAC) transplanted mice models was carried out to evaluate the in-vivo antioxidant and anticancer potential of MEAC. The major bioactive nutraceutical compound present in MEAC was isolated by bioactivity-guided fractionation. MEAC showed significant in vitro antioxidant activity. Further, MEAC promoted cytotoxicity against HT-29 cells by activating caspase-3 dependent apoptotic pathway with a cell cycle arrest at the G1/S phase and subsequent down regulation of COX-2 pathway. The potential antitumor activity of MEAC was further confirmed in EAC tumor bearing mice models in which treatment with MEAC increased the levels of antioxidant enzymes, improved the hematological profile towards normal and also augmented the life span of tumor bearing mice. ß-sitosterol isolated from ACW induces anticancer activity via caspase-dependent pathway. Our study confirmed the antioxidant and anticancer activities of ACW, which proposes the medicinal importance of this plant as a preventive and supportive therapy for arising tumors.

Genetic diversity and population structure of Amorphophallus albus, a plant species with extremely small populations (PSESP) endemic to dry-hot valley of Jinsha River.

BACKGROUND: Amorphophallus albus P. Y. Liu & J. F. Chen (Araceae) is a plant species with extremely small populations (PSESP) and an important economic crop endemic to dry-hot valleys along the Jinsha River. In order to gain information for sustaining the development and conservation of A. albus, we studied the genetic diversity and population structure of this species using microsatellite markers (SSR). In this study, we analysed 364 individuals belonging to 24 populations, including four wild populations and three ex-situ cultivated populations, collected in the provinces Yunnan, Sichuan and Hubei. RESULTS: The population genetic analyses indicated that A. albus possesses moderate genetic diversity with the percentage of polymorphic loci (PPL) from 69.23 to 100%, an expected heterozygosity (He) of 0.504 and an average Shannon's Information Index (I) 0.912. Analysis of molecular variance (AMOVA) indicated that most of the variance (71%) resided within populations and the estimated gene flow (Nm) was 0.61. The results of UPGMA cluster tree, STRUCTURE analyses together with the Mantel test (R2 = 0.352, P < 0.01) indicated that geographically closely located populations are clustered together with some exceptions. CONCLUSIONS: Our results showed that A. albus still possesses moderate genetic variation in most of the studied populations, and for now, most cultivated populations were naturally distributed but still some reintroduction exists. For sustaining the present genetic variation, some protections measures are necessary for the wild populations and also for the cultivated ones with high genetic diversity.

Amorphophallus konjac anthracnose caused by Colletotrichum siamense in China.

AIMS: Amorphophallus konjac is an important commercial crop grown in China because it is the only plant species which is rich in glucomannan concentration. Recently, an outbreak of anthracnose (incidence ranging from 10-15%) was observed in a field survey conducted from June to August 2018. This study aims to identify the causal agent of A. konjac anthracnose. METHODS AND RESULTS: The pathogen was isolated on potato dextrose agar (PDA) medium. The fungal colony on PDA was greyish to dark grey. Conidia were falcate, one-celled and hyaline. Based on the micro-morphological and cultural characteristics, the pathogen was identified as Colletotrichum sp. blast search and phylogenetic analysis of the ITS, GAPDH, CHS1, ACT, CAL and TUB2 genes revealed the pathogen as Colletotrichum siamense. Koch's postulates were conducted on 2-month konjac leaves with conidial suspension. Development of typical anthracnose disease was recorded 5 days after inoculation and the pathogen's identity was confirmed by re-isolation and molecular identification. CONCLUSIONS: Amorphophallus konjac anthracnose was caused by C. siamense in China. SIGNIFICANCE AND IMPACT OF THE STUDY: Identification of causal agent of A. konjac anthracnose will be helpful in designing effective disease control strategies.

Taro koji of Amorphophallus konjac enabling hydrolysis of konjac polysaccharides to various biotechnological interest.

Due to the indigestibility, utilization of konjac taro, Amorphophallus konjac has been limited only to the Japanese traditional konjac food. Koji preparation with konjac taro was examined to utilize konjac taro as a source of utilizable carbohydrates. Aspergillus luchuensis AKU 3302 was selected as a favorable strain for koji preparation, while Aspergillus oryzae used extensively in sake brewing industry was not so effective. Asp. luchuensis grew well over steamed konjac taro by extending hyphae with least conidia formation. Koji preparation was completed after 3-day incubation at 30°C. D-Mannose and D-glucose were the major monosaccharides found in a hydrolyzate giving the total sugar yield of 50 g from 100 g of dried konjac taro. An apparent extent of konjac taro hydrolysis at 55°C for 24 h seemed to be completed. Since konjac taro is hydrolyzed into monosaccharides, utilization of konjac taro carbohydrates may become possible to various products of biotechnological interest.

No Effect of Glucomannan on Body Weight Reduction in Children and Adolescents with Overweight and Obesity: A Randomized Controlled Trial.

OBJECTIVE: To assess the efficacy of water-soluble dietary fiber, glucomannan supplementation, on the body mass index (BMI) in children with overweight or obesity. STUDY DESIGN: In this randomized, double-blind, placebo-controlled trial, we enrolled 96 children aged 6-17 years with overweight or obesity based on the World Health Organization growth criteria (>+1 SD or >+2 SD, respectively). Participants were assigned to receive glucomannan or placebo (maltodextrin), both at a dose of 3 g/d for 12 weeks and were followed up for the next 12 weeks. Concomitant care included dietary and lifestyle advice. The primary outcome was the difference in the BMI-for-age z score change between the groups at 12 weeks. RESULTS: Compared with the placebo, glucomannan had no effect on the BMI-for-age z score at 12 weeks (mean difference: 0.0, 95% CI -0.1 to 0.1). Compared with the placebo, the glucomannan group had lower total and low-density lipoprotein cholesterol concentrations at 12 weeks. In addition, the blood pressure was greater at 12 weeks (systolic) and at 24 weeks (diastolic) in the glucomannan group. No differences between the groups in adverse events and other secondary outcomes were observed. CONCLUSIONS: Glucomannan supplementation compared with placebo had no effect on weight reduction in children with overweight and obesity. TRIAL REGISTRATION: ClinicalTrials.govNCT02280772.

Mucosal Injury From Calcium Oxalate Crystals Resembling Anaphylaxis and Angioedema.

BACKGROUND: There are 215 families of plants that contain insoluble needle-shaped calcium oxalate crystals on the surface of their tissues. Upon mucosal contact, injury can cause extreme pain, soft-tissue swelling, salivation, dysphagia, and even aphonia. This presentation can resemble angioedema or anaphylaxis. CASE REPORT: A 55-year-old Asian female presented to the emergency department complaining of oral pain, swelling, and numbness. Her family reported that she began to experience sharp pain of the tongue and lips immediately after eating "elephant root." Physical examination revealed a patient sitting in an upright position, leaning forward with pooling secretions. She had few lingual petechiae, a subtle diffuse erythema, and mild edema of the lower lip. Due to pain, she was unable to speak and swallow. Her vitals remained within normal limits. The patient was taking lisinopril for hypertension. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: Injury by calcium oxalate crystals is a relatively common occurrence that will present to the emergency department. Although most exposures are benign, patients can develop critical illness, requiring emergent therapies and airway management. Due to the nature of presentation, exposure can easily be misdiagnosed as anaphylaxis or hereditary and drug-induced angioedema. Severe pain and the temporal relationship to plant ingestion distinguish insoluble calcium oxalate crystal exposure from these alternative causes of angioedema. There is minimal evidence-based data evaluating treatment of these injuries. Standard treatment regimen includes a local anesthetic, corticosteroids, opioids, and antihistaminergic agents. Given the relative low cost, ease of administration, and benign adverse effect profile, sodium bicarbonate rinse may have a role as an adjunct therapy, however, research is needed.

Analysis of bacterial communities in rhizosphere soil of continuously cropped healthy and diseased konjac.

The bacterial community and diversity in healthy and diseased konjac rhizosphere soils with different ages of continuous cropping were investigated using next-generation sequencing. The results demonstrated that the number of years of continuous cropping significantly altered soil bacterial community and diversity. Soil bacterial Shannon diversity index and Chao 1 index decreased with the increasing cropping years of konjac. After 1 year of cropping, the soil exhibited the highest bacterial relative abundance and diversity. Of the 44 bacterial genera (relative abundance ratio of genera greater than 0.3%), 14 were significantly affected by the duration of continuous cropping and plant status. With increasing continuous cropping, Alicyclobacillus decreased, while Achromobacter, Lactobacillus, Kaistobacter, Rhodoplanes increased after 3 years continuous cropping. Continuous cropping altered the structure and composition of the soil bacterial community, which led to the reduction in the beneficial bacteria and multiplication of harmful bacteria. These results will improve our understanding of soil microbial community regulation and soil health maintenance in konjac farm systems.

Reconstruction of molecular phylogeny of closely related Amorphophallus species of India using plastid DNA marker and fingerprinting approaches.

Plastid DNA markers sequencing and DNA fingerprinting approaches were used and compared for resolving molecular phylogeny of closely related, previously unexplored Amorphophallus species of India. The utility of individual plastid markers namely rbcL, matK, trnH-psbA, trnLC-trnLD, their combined dataset and two fingerprinting techniques viz. RAPD and ISSR were tested for their efficacy to resolves Amorphophallus species into three sections specific clades namely Rhaphiophallus, Conophallus and Amorphophallus. In the present study, sequences of these four plastid DNA regions as well as RAPD and ISSR profiles of 16 Amorphophallus species together with six varieties of two species were generated and analyzed. Maximum likelihood and Bayesian Inference based construction of phylogenetic trees indicated that among the four plastid DNA regions tested individually and their combined dataset, rbcL was found best suited for resolving closely related Amorphophallus species into section specific clades. When analyzed individually, rbcL exhibited better discrimination ability than matK, trnH-psbA, trnLC-trnLD and combination of all four tested plastid markers. Among two fingerprinting techniques used, the resolution of Amorphophallus species using RAPD was better than ISSR and combination of RAPD +ISSR and in congruence with resolution based on rbcL.