The endophytic fungal community plays a crucial role in the resistance of host plants to necrotic bacterial pathogens.

Konjac species (Amorphophallus spp.) are the only plant species in the world that are rich in a large amount of konjac glucomannan (KGM). These plants are widely cultivated as cash crops in tropical and subtropical countries in Asia, including China. Pectobacterium carotovorum subsp. carotovorum (Pcc) is one of the most destructive bacterial pathogens of konjac. Here, we analyzed the interactions between Pcc and susceptible and resistant konjac species from multiple perspectives. At the transcriptional and metabolic levels, the susceptible species A. konjac and resistant species A. muelleri exhibit similar molecular responses, activating plant hormone signaling pathways and metabolizing defense compounds such as phenylpropanoids and flavonoids to resist infection. Interestingly, we found that Pcc stress can lead to rapid recombination of endophytic microbial communities within a very short period (96 h). Under conditions of bacterial pathogen infection, the relative abundance of most bacterial communities in konjac tissue decreased sharply compared with that in healthy plants, while the relative abundance of some beneficial fungal communities increased significantly. The relative abundance of Cladosporium increased significantly in both kinds of infected konjac compared to that in healthy plants, and the relative abundance in resistant A. muelleri plants was greater than that in susceptible A. konjac plants. Among the isolated cultivable microorganisms, all three strains of Cladosporium strongly inhibited Pcc growth. Our results further elucidate the potential mechanism underlying konjac resistance to Pcc infection, highlighting the important role of endophytic microbial communities in resisting bacterial pathogen infections, especially the more direct role of fungal communities in inhibiting pathogen growth.

Phenylpropane biosynthesis and alkaloid metabolism pathways involved in resistance of Amorphophallus spp. against soft rot disease.

Soft rot of konjac (Amorphophallus spp.) is a devastating disease caused by the bacterium Pectobacterium carotovorum subsp. carotovorum (Pcc) with serious adverse effects on plantation development, corm quality and crop yield due to the current lack of effective control measures. The main objective of the present study was to elucidate the mechanisms underlying plant resistance to soft rot disease. A combination of transcriptomic and metabolomic analyses demonstrated significant enrichment of differentially expressed genes (DEG) and differentially accumulated metabolites (DAM) associated with plant hormones, phenylpropanoid biosynthesis and, in particular, alkaloid metabolism, in Amorphophallus muelleri following Pcc infection compared with A. konjac, these data implicate alkaloid metabolism as the dominant mechanism underlying disease resistance of A. muelleri. Quantitative real-time polymerase chain reaction analysis further revealed involvement of PAL, CYP73A16, CCOAOMT1, RBOHD and CDPK20 genes in the response of konjac to Pcc. Analysis of the bacteriostatic activities of total alkaloid from A. muelleri validated the assumption that alkaloid metabolism positively regulates disease resistance of konjac. Our collective results provide a foundation for further research on the resistance mechanisms of konjac against soft rot disease.

The complete chloroplast genome sequence of Amorphophallus konjac (Araceae) from Yunnan, China and its phylogenetic analysis in the family Araceae.

This work determined and analyzed the complete chloroplast genome sequence of Amorphophallus konjac K. Koch ex N.E.Br 1858 from Yunnan, China. The genome size was 167,470 bp, of which contains a large single-copy region (LSC 93,443 bp), a small single-copy region (SSC 21,575 bp), and a pair of inverted repeat regions (IR 26,226 bp). The chloroplast genome has 131 genes, including 86 protein-coding genes, 37 tRNAs, and eight rRNAs. A previous study reported deletion of accD, psbE, and trnG-GCC genes in the A. konjac chloroplast genome. Our study supports the conservative structure of A. konjac and does not support the gene deletion mentioned above. Phylogenetic analysis indicated that A. konjac shares a close relationship with another A. konjac (collected from Guizhou) and A. titanium by forming a clade in the genus Amorphophallus. Our results provide some useful information to the evolution of the family Araceae.

Comparative metabolomics analysis reveals dynamic changes in carbohydrate profiles of corms during the "relay growth" of konjac (Amorphophallus muelleri).

The type and content of carbohydrates in konjac corms are an essential factors in determining the quality of konjac; however, the pattern of carbohydrate changes and the mechanism regulating the development of mother and daughter corms in the "relay growth" process of Amorphophallus muelleri remain unclear. This study aimed to investigate changes in corm carbohydrates during the growth cycle of A. muelleri and to compare the carbohydrate composition and the expression of related genes between mother and daughter corms. Integrated metabolome and RNA-seq analyses identified 37 differential metabolites as well as 8074 genes that were differentially expressed between mother and daughter corms, the majority of which were involved in starch and sucrose metabolism. More than 80% of the differential metabolites, including sucrose and starch, tended to accumulate in the mother corms; however, konjac glucomannan (KGM), as one of the most important carbohydrates and its major component of the corm, accumulated in higher amounts in the daughter corms. In addition, the expression of invertase and alpha-amylase that promote the breakdown of sucrose and starch was 351.78- and 15.63-fold higher, respectively, in the daughter corm, whereas that of the starch synthesis gene AkWAXY was only 0.096 times as high as in the mother corms. Furthermore, the level of cellulose synthase-like protein G, which promotes KGM synthesis, was 3.85 times higher in daughter corms compared to mother corms. Thus, we inferred that the daughter and mother corms had two distinct carbohydrate utilization strategies. This study provides insights into temporal changes in carbohydrates during the growth cycle of A. muelleri.

Amorphophallus muelleri activates ferulic acid and phenylpropane biosynthesis pathways to defend against Fusarium solani infection.

Amorphophallus sp. is an economically important crop for rural revitalization in southwest China. However, Fusarium solani often infects Amorphophallus sp. corms during storage, damaging the corm quality and affecting leaf elongation and flowering in the subsequent crop. In this study, the mechanism of resistance to F. solani was investigated in the leaf bud and flower bud corms of Amorphophallus muelleri through transcriptome and metabolome analyses. A total of 42.52 Gb clean reads and 1,525 metabolites were detected in a total of 12 samples including 3 samples each of disease-free leaf bud corms (LC), leaf bud corms inoculated with F. solani for three days (LD), disease-free flower bud corms (FC), and flower bud corms inoculated with F. solani for three days (FD). Transcriptome, metabolome, and conjoint analyses showed that 'MAPK signal transduction', 'plant-pathogen interaction', 'plant hormone signal transduction', and other secondary metabolite biosynthesis pathways, including 'phenylpropane biosynthesis', 'arachidonic acid metabolism', 'stilbene, diarylheptane and gingerolin biosynthesis', and 'isoquinoline alkaloids biosynthesis', among others, were involved in the defense response of A. muelleri to F. solani. Ultimately, the expression of six genes of interest (AmCDPK20, AmRBOH, AmWRKY33, Am4CL, Am POD and AmCYP73A1) was validated by real-time fluorescence quantitative polymerase chain reaction, and the results indicated that these genes were involved in the response of A. muelleri to F. solani. Ferulic acid inhibited the growth of F. solani, reducing the harm caused by F. solani to A. muelleri corms to a certain extent. Overall, this study lays a strong foundation for further investigation of the interaction between A. muelleri and F. solani, and provides a list of genes for the future breeding of F. solani-resistant A. muelleri cultivars.

First report of Athelia rolfsii associated with southern blight disease of Amorphophallus muelleri in China.

Amorphophallus muelleri, known as konjac, is widely used in the biomedicine and food processing due to its richness in glucomannan. Between the years of 2019 to 2022, severe outbreaks of southern blight on Am. muelleri were observed during August and September in the main planting region of Mile city. The average disease incidence was 20%, resulted in 15.3% of economic losses in approximately 10,000 m2. Infected plants showed wilting and rotting and were covered with white dense mats of mycelia and sclerotia on both petiole base and tubers. Am. muelleri petiole base covered with mycelial mats were collected for pathogen isolation. The infected tissues (n=20) were washed with sterile water and surface disinfected with 75% alcohol for 60 seconds, rinsed three times with sterile water, cultured on rose bengal agar (RBA) and incubated at 27 ℃ for two days (Adre et al. 2022). Individual hyphae were transferred to new RBA plates and incubated at 27 ℃ for 15 days to obtain purified cultures. Five representative isolates were subsequently obtained and exhibited identical morphological characteristics. All isolates produced dense, cotton-white aerial mycelia and had a daily growth rate of 1.6 ± 0.2 mm (n=5). After 10 days, all isolates formed sclerotia in spherical (diameter range 1.1 to 3.5 mm, aver. 2.0 ± 0.5 mm; n=30) and irregular shapes. The number of sclerotia per plate ranged from 58 to 113 (aver=82; n=5). These sclerotia were initially white and gradually turned brown as they matured. A representative isolate (17B-1) was selected for molecular identification and the translation elongation factor (TEF, 480 nt.), internal transcribed spacer (ITS, 629 nt.), large subunit (LSU, 922 nt.), and small subunit (SSU, 1016 nt.) regions were amplified with the primers EF595F/EF1160R (Wendland and Kothe 1997), ITS1/ITS4 (Utama et al. 2022), NS1/NS4, and LROR/LR5 (Moncalvo et al. 2000), respectively. The ITS (GenBank accession no. OP658949), LSU (OP658955), SSU (OP658952), and TEF (OP679794) sequences were 99.19%, 99.78%, 99.31%, and 99.58% similar to those of At. rolfsii isolates (MT634388, MT225781, MT103059, and MN106270, respectively). Thus, the fungus represented by isolate 17B-1 was identified as At. rolfsii, corroborating the identification of Sclerotium rolfsii Sacc., the anamorph, based on cultural and morphological features. Pathogenicity tests were performed on six-month-old asymptomatic Am. muelleri plants (n=30) grown in pots with sterile soil in a greenhouse at 27 °C and 80% relative humidity. The petiole base was scratched with a sterile blade and 20 plants were inoculated by placing a 5 mm2 mycelial plug of five-day-old isolate 17B-1 on the wound. Sterile RBA plugs were used on 10 wounded control plants. After 12 days, all inoculated plants exhibited symptoms similar to those observed in the field, while the control plants showed no symptoms. The morphological and molecular identification of the fungus reisolated from inoculated petioles confirmed its identity as At. Rolfsii, fulfilling Koch's postulates. S. rolfsii was first reported on Am. campanulatus in India (Sarma et al. 2002). As At. rolfsii causes konjac diseases in all Amorphophallus growing areas (Pravi et al. 2014), the importance of At. rolfsii as an endemic pathogen of Am. muelleri in China needs to be recognized, and its prevalence should be determined as a first step to managing this disease.

Different Response Mechanisms of Rhizosphere Microbial Communities in Two Species of Amorphophallus to Pectobacterium carotovorum subsp. carotovorum Infection.

Soft rot is a widespread, catastrophic disease caused by Pectobacterium carotovorum subsp. carotovorum (Pcc) that severely damages the production of Amorphophallus spp. This study evaluated the rhizosphere bacterial and fungal communities in Pcc-infected and uninfected plants of two species of Amorphophallus, A. muelleri and A. konjac. Principal component analysis showed that the samples formed different clusters according to the Pcc infection status, indicating that Pcc infection can cause a large number of changes in the bacterial and fungal communities in the Amorphophallus spp. rhizosphere soil. However, the response mechanisms of A. muelleri and A. konjac are different. There was little difference in the overall microbial species composition among the four treatments, but the relative abundances of core microbiome members were significantly different. The relative abundances of Actinobacteria, Chloroflexi, Acidobacteria, Firmicutes, Bacillus, and Lysobacter were lower in infected A. konjac plants than in healthy plants; in contrast, those of infected A. muelleri plants were higher than those in healthy plants. For fungi, the relative abundances of Ascomycota and Fusarium in the rhizosphere of infected A. konjac plants were significantly higher than those of healthy plants, but those of infected A. muelleri plants were lower than those of healthy plants. The relative abundance of beneficial Penicillium fungi was lower in infected A. konjac plants than in healthy plants, and that of infected A. muelleri plants was higher than that of healthy plants. These findings can provide theoretical references for further functional research and utilization of Amorphophallus spp. rhizosphere microbial communities in the future.

A high trans-zeatin nucleoside concentration in corms may promote the multileaf growth of Amorphophallus muelleri.

Amorphophallus muelleri has a multileaf growth pattern different from that of other konjacs; however, the hormonal mechanism underlying this phenomenon is not clear. In this study, the levels of hormones closely related to the sprouting of the axillary bud, including five types of cytokinins, indole-3-acetic acid (IAA) and abscisic acid (ABA) were measured. In the second leaf sprouting stage, the content of trans-zeatin riboside (tZR) in corms increased more than 5000-fold over that in the dormancy period. Surprisingly, although the expression of CYP735A1 and CYP735A2, which synthesize the precursors for tZR was elevated at the second leaf sprouting stage, the expression of IPTs, which have key roles in cytokinin biosynthesis, did not change significantly. In addition, most cytokinin contents in leaves during the same period were significantly lower than those in corms. We speculate that the high cytokinin contents in the corms may not biosynthesized de novo in corms. In addition, the IAA content in the corms also considerably increased during the second leaf sprouting stage. Indole-3-acetaldehyde oxidase (AO1) and auxin efflux carrier PIN1A, presented relatively high expression levels in the same period. In contrast, ABA content, and the expression of NCED1, a rate-limiting enzyme in ABA biosynthesis, were suppressed at the second leaf sprouting stage. It is worth mentioning that N6-(Δ2-isopentenyl) adenosine (iP)-type cytokinins have a high content in corms in the dormant period that significantly decreases after the first leaf sprouting stage, which is completely different from the trend of tZR. By treating dormant corms with iP, the percentage of multibud plants increased, and the growth performance in terms of bud and root length was significantly higher than those of the control. This implies that iP-type cytokinins tend to play a role in promoting first seedling sprouting. Furthermore, there was a remarkable increase of the IAA content in both corms and roots under iP treatment but an inhibitory effect in buds. We speculate that the increase in the IAA content induced by iP is tissue specific. These results will assist in the understanding of the role of hormones, especially cytokinins, in the multileaf growth type of konjac.

Dynamic changes in the endophytic bacterial community during maturation of Amorphophallus muelleri seeds.

The seed microbiota is considered to be the starting point of the accumulation of plant microbiota, which is conducive to the preservation and germination of seeds and the establishment and development of seedlings. Our understanding of the colonization and migration dynamics of microbial taxa during seed development and maturation is still limited. This study used 16S rRNA high-throughput sequencing to investigate the dynamic changes in the composition and diversity of the endophytic bacterial community during maturation of Amorphophallus muelleri seeds. The results showed that as seeds matured (green to red), the Shannon index of their endophytic bacterial community first decreased and then increased, and the ACE and Chao1 indices of the endophytic bacterial community decreased gradually. The Shannon, ACE, and Chao1 indices of the endophytic bacterial community in the seed coat first decreased and then increased. Principal coordinate analysis of the bacterial communities revealed that the seed coat at different maturity stages showed significantly distinct bacterial communities and formed different clusters according to maturity stage. The bacterial communities of green and red seeds showed a clear separation, but they both overlapped with those of yellow seeds, indicating that some core taxa were present throughout seed maturation, but their relative abundance was dynamically changing. As the seeds grew more mature, the relative abundance of some bacterial communities with plant growth-promoting traits and others correlated with plant resistance (e.g., Burkholderia-Caballeronia-Paraburkholderia, Bacillus, Pseudomonas, Bradyrhizobium, Streptomyces) tended to increase and peaked in fully mature seeds and seed coats. The endophytic bacterial community of A. muelleri seeds seems to be driven by the seed maturation state, which can provide a theoretical basis for a comprehensive understanding of the assembly process of the microbial community during A. muelleri seed maturation.

Transcriptomic and metabolomic changes triggered by Macrosiphum rosivorum in rose (Rosa longicuspis).

BACKGROUND: Rose is one of the most popular flowers in the wold. Its field growth and quality are negatively affected by aphids. However, the defence mechanisms used by rose plants against aphids are unclear. Therefore, to understand the defence mechanism of rose under aphid stress, transcriptome and metabolome techniques were used to investigate the regulation mechanism in R. longicuspis infected with M. rosivorum. RESULT: In our study, after inoculation with M. rosivorum, M. rosivorum quickly colonized R. longicuspis. A total of 34,202 genes and 758 metabolites were detected in all samples. Under M. rosivorum stress, R. longicuspis responded by MAPK cascades, plant hormone signal transduction pathway activation, RlMYBs and RlERFs transcription factors expression and ROS production. Interestingly, the 'brassinosteroid biosynthesis' pathway was significantly enriched in A3 d-vs.-A5 d. Further analysis showed that M. rosivorum induced the biosynthesis of secondary metabolites such as terpenoids, tannins and phenolic acids, among others. Importantly, the 'glutathione metabolic' and 'glucosinolate biosynthesis' pathways were significantly enriched, which involved in the rose against aphids. CONCLUSION: Our study provides candidate genes and metabolites for Rosa defence against aphids. This study provides a theoretical basis for further exploring the molecular regulation mechanism of rose aphid resistance and aphid resistance breeding in the future.

RcTGA1 and glucosinolate biosynthesis pathway involvement in the defence of rose against the necrotrophic fungus Botrytis cinerea.

BACKGROUND: Rose is an important economic crop in horticulture. However, its field growth and postharvest quality are negatively affected by grey mould disease caused by Botrytis c. However, it is unclear how rose plants defend themselves against this fungal pathogen. Here, we used transcriptomic, metabolomic and VIGS analyses to explore the mechanism of resistance to Botrytis c. RESULT: In this study, a protein activity analysis revealed a significant increase in defence enzyme activities in infected plants. RNA-Seq of plants infected for 0 h, 36 h, 60 h and 72 h produced a total of 54 GB of clean reads. Among these reads, 3990, 5995 and 8683 differentially expressed genes (DEGs) were found in CK vs. T36, CK vs. T60 and CK vs. T72, respectively. Gene annotation and cluster analysis of the DEGs revealed a variety of defence responses to Botrytis c. infection, including resistance (R) proteins, MAPK cascade reactions, plant hormone signal transduction pathways, plant-pathogen interaction pathways, Ca2+ and disease resistance-related genes. qPCR verification showed the reliability of the transcriptome data. The PTRV2-RcTGA1-infected plant material showed improved susceptibility of rose to Botrytis c. A total of 635 metabolites were detected in all samples, which could be divided into 29 groups. Metabonomic data showed that a total of 59, 78 and 74 DEMs were obtained for T36, T60 and T72 (T36: Botrytis c. inoculated rose flowers at 36 h; T60: Botrytis c. inoculated rose flowers at 60 h; T72: Botrytis c. inoculated rose flowers at 72 h) compared to CK, respectively. A variety of secondary metabolites are related to biological disease resistance, including tannins, amino acids and derivatives, and alkaloids, among others; they were significantly increased and enriched in phenylpropanoid biosynthesis, glucosinolates and other disease resistance pathways. This study provides a theoretical basis for breeding new cultivars that are resistant to Botrytis c. CONCLUSION: Fifty-four GB of clean reads were generated through RNA-Seq. R proteins, ROS signalling, Ca2+ signalling, MAPK signalling, and SA signalling were activated in the Old Blush response to Botrytis c. RcTGA1 positively regulates rose resistance to Botrytis c. A total of 635 metabolites were detected in all samples. DEMs were enriched in phenylpropanoid biosynthesis, glucosinolates and other disease resistance pathways.