Ancient bayberry increased stress resistance by enriching tissue-specific microbiome and metabolites.

The ancient bayberry demonstrates superior resistance to both biotic and abiotic stresses compared to cultivated bayberry, yet the underlying mechanisms remain largely unexplored. This study investigates whether long-term bayberry cultivation enhances stress resistance through modulation of tissue-specific microbes and metabolites. Employing microbiome amplicon sequencing alongside untargeted mass spectrometry analysis, we scrutinize the role of endosphere and rhizosphere microbial communities and metabolites in shaping the differential resistance observed between ancient and cultivated bayberry trees. Our findings highlight the presence of core microbiome and metabolites across various bayberry tissues, suggesting that the heightened resistance of ancient bayberry may stem from alterations in rhizosphere and endosphere microbial communities and secondary metabolites. Specifically, enrichment of Bacillus in roots and stems, Pseudomonas in leaves, and Mortierella in rhizosphere soil of ancient bayberry was noted. Furthermore, correlation analysis underscores the significance of enriched microbial species in enhancing ancient bayberry's resistance to stresses, with elevated levels of resistance-associated metabolites such as beta-myrcene, benzothiazole, L-glutamic acid, and gamma-aminobutyric acid identified through GC-MS metabolomics analysis. The beneficial role of these resistance-associated metabolites was further elucidated through assessment of their promotive and allelopathic effects, as well as their phytostatic and antioxidant functions in lettuce plants. Ultimately, our study delves into the intrinsic reasons behind the greater resistance of ancient bayberry to biotic and abiotic stresses by evaluating the impact of long-term planting on the microbial community and metabolites in the bayberry endosphere and rhizosphere, shedding light on the complex dynamics of host-microbial interactions.

Multiple mycoviruses identified in Pestalotiopsis spp. from Chinese bayberry.

BACKGROUND: Chinese bayberry (Myrica rubra) is a subtropical fruit crop widely grown in southern China. Twig dieback is a disease of Chinese bayberry caused by Pestalotiopsis spp. and results in great economic losses to Chinese bayberry production. A virus survey was conducted in the population of Pestalotiopsis spp. infecting M. rubra in China. We explored the viral diversity in Pestalotiopsis spp., which may provide resources for further development as biocontrol agents of twig dieback. METHODS: Strains of Pestalotiopsis spp. were isolated from diseased twigs of M. rubra, and cultured on potato dextrose agar for RNA extraction. The total RNA of each strain was extracted, mixed, and used for RNA sequencing. The resulting sequences were deduplicated, annotated, and then used for phylogenetic analysis. RESULTS: Seven novel viruses were characterized from 59 isolates of M. rubra collected from 14 localities in China. Based on the phylogenetic analysis, these viruses were classified into five viral families/orders, Botourmiaviridae, Mitoviridae, Partitiviridae, Tymovirales and Bunyavirales, and one virus, Pestalotiopsis negative-stranded RNA virus 1, which likely belongs to a new viral family. CONCLUSIONS: Metatranscriptomics analysis showed the presence of various mycoviruses in Pestalotiopsis spp. isolated from M. rubra in China. The genomes of eight putative viruses were identified, seven of which were nearly full-length. Some of these viruses of Pestalotiopsis spp. may have the potential for the biological control of twig dieback of M. rubra.

Frankia asymbiotica sp. nov., a non-infective actinobacterium isolated from Morella californica root nodule.

The taxonomic status of strain M16386T, a nitrogen-fixing but non-nodulating isolate from Morella californica, was established on the basis of a polyphasic approach. The strain grows as branched hyphae, with vesicles and non-motile productive multilocular sporangia. It metabolizes short fatty acids, TCA cycle intermediates and carbohydrates as carbon sources, and fixes nitrogen in the absence of combined nitrogen source in the growth media. Chemotaxonomic traits of strain M16386T are consistent with its affiliation to the genus Frankia. The characteristic diamino acid in the cell wall is meso-diaminopimelic acid. Strain M16386T contains phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol, glycophospholipid and phospholipid as polar lipids; MK-9(H4) and MK-9(H6) as the predominant menaquinones; iso-C16 : 0 and C17 : 1ω8c as major fatty acids; and galactose, glucose, mannose, rhamnose and ribose as whole-cell sugars. Strain M16386T showed 98.2 % 16S rRNA gene sequence similarity with its closest phylogenetic neighbour, Frankia inefficaxDSM 45817T. Based on these results, strain M16386T (=DSM 100626T=CECT 9040T) is designated the type strain of a novel species of the genus Frankia,for which the name Frankia asymbiotica sp. nov. is proposed.

Micromonospora is a normal occupant of actinorhizal nodules.

Actinorhizal plants have been found in eight genera belonging to three orders (Fagales, Rosales and Cucurbitales). These all bear root nodules inhabited by bacteria identified as the nitrogen-fixing actinobacterium Frankia. These nodules all have a peripheral cortex with enlarged cells filled with Frankia hyphae and vesicles. Isolation in pure culture has been notoriously difficult, due in a large part to the growth of fast-growing contaminants where, it was later found, Frankia was slow-growing. Many of these contaminants, which were later found to be Micromonospora, were obtained from Casuarina and Coriaria. Our study was aimed at determining if Micromonospora were also present in other actinorhizal plants. Nodules from Alnus glutinosa, Alnus viridis, Coriaria myrtifolia, Elaeagnus x ebbingei, Hippophae rhamnoides, Myrica gale and Morella pensylvanica were tested and were all found to contain Micromonospora isolates. These were found to belong to mainly three species: Micromonospora lupini, Micromonospora coriariae and Micromonospora saelicesensis. Micromonospora isolates were found to inhibit some Frankia strains and to be innocuous to other strains.

Amplicon restriction patterns associated with nitrogenase activity of root nodules for selection of superior Myrica seedlings.

Trees of Myrica sp. grow abundantly in the forests of Meghalaya, India. These trees are actinorhizal and harbour nitrogen-fixing Frankia in their root nodules and contribute positively towards the enhancement of nitrogen status of forest areas. They can be used in rejuvenation of mine spoils and nitrogen-depleted fallow lands generated due to slash and burn agriculture practiced in the area. We have studied the association of amplicon restriction patterns (ARPs) of Myrica ribosomal RNA gene and internal transcribed spacer (ITS) region and nitrogenase activity of its root nodules. We found that ARPs thus obtained could be used as markers for early screening of seedlings that could support strains of Frankia that fix atmospheric nitrogen more efficiently.

Transcriptomes of Frankia sp. strain CcI3 in growth transitions.

BACKGROUND: Frankia sp. strains are actinobacteria that form N2-fixing root nodules on angiosperms. Several reference genome sequences are available enabling transcriptome studies in Frankia sp. Genomes from Frankia sp. strains differ markedly in size, a consequence proposed to be associated with a high number of indigenous transposases, more than 200 of which are found in Frankia sp. strain CcI3 used in this study. Because Frankia exhibits a high degree of cell heterogeneity as a consequence of its mycelial growth pattern, its transcriptome is likely to be quite sensitive to culture age. This study focuses on the behavior of the Frankia sp. strain CcI3 transcriptome as a function of nitrogen source and culture age. RESULTS: To study global transcription in Frankia sp. CcI3 grown under different conditions, complete transcriptomes were determined using high throughput RNA deep sequencing. Samples varied by time (five days vs. three days) and by culture conditions (NH4+ added vs. N2 fixing). Assembly of millions of reads revealed more diversity of gene expression between five-day and three-day old cultures than between three day old cultures differing in nitrogen sources. Heat map analysis organized genes into groups that were expressed or repressed under the various conditions compared to median expression values. Twenty-one SNPs common to all three transcriptome samples were detected indicating culture heterogeneity in this slow-growing organism. Significantly higher expression of transposase ORFs was found in the five-day and N2-fixing cultures, suggesting that N starvation and culture aging provide conditions for on-going genome modification. Transposases have previously been proposed to participate in the creating the large number of gene duplication or deletion in host strains. Subsequent RT-qPCR experiments confirmed predicted elevated transposase expression levels indicated by the mRNA-seq data. CONCLUSIONS: The overall pattern of gene expression in aging cultures of CcI3 suggests significant cell heterogeneity even during normal growth on ammonia. The detection of abundant transcription of nif (nitrogen fixation) genes likely reflects the presence of anaerobic, N-depleted microsites in the growing mycelium of the culture, and the presence of significantly elevated transposase transcription during starvation indicates the continuing evolution of the Frankia sp. strain CcI3 genome, even in culture, especially under stressed conditions. These studies also sound a cautionary note when comparing the transcriptomes of Frankia grown in root nodules, where cell heterogeneity would be expected to be quite high.

Effect of hot air treatment on postharvest mould decay in Chinese bayberry fruit and the possible mechanisms.

The effect of hot air treatment (HAT) on reducing natural fungal decay and green mould decay caused by Leptographium abietinum on postharvest Chinese bayberry fruit and the possible mechanisms were investigated. Freshly harvested Chinese bayberry fruit were firstly pretreated with hot air at 36-60 degrees C for 1-3h, and then stored at 20 degrees C for 3d or at 1 degrees C for 12d to investigate the optimum condition of hot air treatment (HAT) for inhibiting decay development. Results demonstrated that HAT at 48 degrees C for 3h was the most effective in reducing natural decay without impairing quality. This treatment also enhanced the resistance of Chinese bayberry fruit against green mould rot caused by L. abietinum and reduced the severity of the disease. The activities of defense-related enzymes including chitinase, beta-1, 3-glucanase, peroxidase and polyphenol oxidase were significantly enhanced by HAT. In addition, the in vitro experiment showed that HAT significantly inhibited spore germination, germ tube elongation and mycelial growth of L. abietinum. These results indicate that HAT can effectively reduce fruit decay possibly by directly inhibiting pathogen growth and indirectly inducing disease resistance.

Differential effects of rare specific flavonoids on compatible and incompatible strains in the Myrica gale-Frankia actinorhizal symbiosis.

Plant secondary metabolites, and specifically phenolics, play important roles when plants interact with their environment and can act as weapons or positive signals during biotic interactions. One such interaction, the establishment of mutualistic nitrogen-fixing symbioses, typically involves phenolic-based recognition mechanisms between host plants and bacterial symbionts during the early stages of interaction. While these mechanisms are well studied in the rhizobia-legume symbiosis, little is known about the role of plant phenolics in the symbiosis between actinorhizal plants and Frankia genus strains. In this study, the responsiveness of Frankia strains to plant phenolics was correlated with their symbiotic compatibility. We used Myrica gale, a host species with narrow symbiont specificity, and a set of compatible and noncompatible Frankia strains. M. gale fruit exudate phenolics were extracted, and 8 dominant molecules were purified and identified as flavonoids by high-resolution spectroscopic techniques. Total fruit exudates, along with two purified dihydrochalcone molecules, induced modifications of bacterial growth and nitrogen fixation according to the symbiotic specificity of strains, enhancing compatible strains and inhibiting incompatible ones. Candidate genes involved in these effects were identified by a global transcriptomic approach using ACN14a strain whole-genome microarrays. Fruit exudates induced differential expression of 22 genes involved mostly in oxidative stress response and drug resistance, along with the overexpression of a whiB transcriptional regulator. This work provides evidence for the involvement of plant secondary metabolites in determining symbiotic specificity and expands our understanding of the mechanisms, leading to the establishment of actinorhizal symbioses.

[Genetic diversity of Frankia in Fujian].

UNLABELLED: There are lots of actinorhizal plants distributed in coastal area and mountains in Fujian province, China. OBJECTIVE: The aim of this study is to describe the genetic diversity of Frankia strains symbiotically associated with several species of Casuarina, Myrica, Alnus and Elaeagnus in Fujian. METHODS: Genomic DNA was extracted from Frankia strains and used as template in PCR with the primers targeting two DNA regions, one in ribosomal operon, and the other in nif D-K gene. PCR products were then digested by using a set of restriction endonucleases to generate the restriction fragment length polymorphism patterns. RESULTS: Except 2 strains nodulating M. rubra and a strain infecting E. oldhami, 17 Frankia strains had an amplified fragment of rrn region in 16S-23S rDNA intergenetic spacer (IGS), 12 Frankia strains nodulating C. equisetifolia, C. cunninghamiana and C. glauca living in 6 geographical origins had high homogeneity and were assigned to one group by clustering analysis, 5 strains from M. rubra and A. cremastogyne were closely related and fell to the other group. All 25 Frankia strains showed a single copy of IGS nif D-K and generated 8 PCR-RFLP patterns while clustering into 3 different groups. Frankia strains nodulating three species of Casuarina in seven different sites had a high degree of genetic similarity to cluster one group. Isolates infecting M. rubra, A. cremastogyne and A. formosana were closely related and belonged to the other cluster, Frankia from E. oldhami owed a quite different cluster. CONCLUSION: These results demonstrated that there is great genetic diversity among Frankia microsymbionts in Fujian.

Genetic diversity of Frankia microsymbionts from the relict species Myrica faya (Ait.) and Myrica rivas-martinezii (S.) in Canary Islands and Hawaii.

In the Western Canary Islands, Myrica faya and Myrica rivas-martinezii (Myricaceae) are phylogenetically close, endemic, actinorhizal species presumed to be remnants either of the European or the African Tertiary floras. Unisolated Frankia strains from field-collected nodules on Tenerife, Gomera, and La Palma Islands were compared by their rrs gene and 16S-23S intergenic spacer (IGS) restriction patterns. To compare the genetic diversity of Frankia strains from within and outside the host's native range, nodules of M. faya field plants were collected both in Canary Islands and in Hawaii, where this species is an exotic invasive. Myrica rivas-martinezii, endemic to the Canary Islands, was sparsely nodulated in the field. Frankia strains harbored in field-collected nodules of M. faya and M. rivas-martinezii belonged to the Elaeagnaceae strains' genetic cluster and exhibited a high degree of diversity. Frankia genotypes were specific to each host species. In the Canary archipelago, we found no relationship between site of collection and Frankia genotype for M. faya. The only exceptions were strains from site 2 in Tenerife, a location with a geological history different from the other sites sampled. Hawaiian and Canarian M. faya strains had no genotypes in common, raising questions concerning the origin of M. faya-infective Frankia in Hawaii. Nodular strains of M. rivas-martinezii from nursery plants were genetically characterized and shown to be divergent from the strains of field-collected nodules and belong to the Alnus-Casuarina strains cluster. This suggests Myrica may have the potential to nodulate with a broader range of Frankia genotypes under artificial conditions than has been detected in field-collected nodules.

Diversity of Frankia strains associated to Myrica gale in Western Europe: impact of host plant (Myrica vs. Alnus) and of edaphic factors.

Myricaceae can be nodulated by a variety of Frankia strains isolated from other actinorhizal families. Consequently, the genus Myrica has been considered to have low specificity with respect to microsymbiont taxa. In contrast to controlled studies of Myrica infectious capacity, field studies in North America have indicated that M. gale symbionts belong to the genetic group of Alnus-infective strains. Myrica gale is the most widely distributed species in the genus so this study focused on describing the genetic diversity of M. gale-nodulating strains from 10 sites in Western Europe across a range of edaphic conditions. When possible, the specificity of M. gale-infective strains was compared with that of Alnus-infective strains from the same sites. Nodular strains from Belgium, France and Spain were characterized using PCR-RFLP of rrs gene and 16S-23S IGS. rrs-RFLP patterns showed a high level of homogeneity among European strains with one dominant genotype. IGS-RFLP patterns revealed the largest inter and intrasite diversity in France. In Belgium, Frankia strains were found to occur in two groups according to soil pH and organic matter characteristics of the sites. European M. gale-infective strains were genetically different from European Alnus and North American M. gale-infective strains indicating the possibility of different pathways of co-evolution among geographically isolated populations.

Arbuscular mycorrhizal fungi colonize decomposing leaves of Myrica parvifolia, M. pubescens and Paepalanthus sp.

Hyphae and vesicles of arbuscular mycorrhizal fungi (AMF) were found within the decomposing leaves of Myrica parvifolia, M. pubescens and Paepalanthus sp. at three montane sites in Colombia. Hyphae, vesicles, and arbuscule-like structures were also found within scale-like leaves of the rhizomes of Paepalanthus sp. The litter found in the vicinity of the roots was divided into three decomposition layers. The highest AMF colonization occurred in the most decomposed leaves, which were in close association with roots. In contrast, there were no differences in AMF colonization of roots present in the different decomposition layers. Colonization of decomposing leaves by AMF did not differ between the two closely related species M. parvifolia and M. pubescens, nor between two sites (Guatavita and Zipacón, Colombia) differing in soil fertility. Occurrence of vesicles in decomposing leaves was correlated with abundant AMF extraradical hyphae among the leaves. We propose that AMF enter decomposing leaves mechanically through vascular tissue. As a consequence, AMF are well positioned to obtain and efficiently recycle mineral nutrients released by decomposer microorganisms before their loss by leaching or immobilization in soil.