Conidia Production of Beauveria Bassiana in Solid Substrate Fermentation Using a Biphasic System

Beauveria bassiana is an important entomopathogenic fungus that is widely used as a bioinsecticide around the world. Conidia production is a critical step in the production of high-quality bioinsecticide. This study investigated three liquid culture mediums and five combinations of solid substrates to enhance conidia production by B. bassiana. The fungus was isolated from infected insects in the cocoa plantation of PT. Perkebunan Nusantara XII in Kediri, East Java, Indonesia. The three culture mediums were malt extract broth (MB), potato dextrose broth (PDB), and yeast and malt extract broth (YMB). Five combinations of solid substrate were used: 100% rice, 100% maize, 75%:25% rice:maize, 50%:50% rice:maize, and 25%:75% rice:maize. The biphasic system was used in this study, in which the fungus was first grown under submerged conditions and then was allowed to conidiate in solid-state conditions. The data showed that PDB was the optimum culture medium to produce blastophore and beauvericin, the active compound that acts as a mycoinsecticide. In the selection test, 100% rice was the optimum solid substrate to produce high amounts of conidia, and the consistency and production tests yielded the same results, with conidia counts of 1.93x109, 1.78x109, and 2.08x109, respectively. In a rice storability test, B. bassiana conidia numbers remained stable for up to 105 days of storage at room temperature. Keywords: Beauveria bassiana, culture medium, solid-substrate, conidia, biphasic syste

Micropropagation of Java Cardamom (Amomum compactum)

Java cardamom (Amomum compactum) is hardly propagated with rhizome without the mother plant.  In vitro culture could overcome the problem through mass propagation for seedling production or other purposes such as genetic material for mutation breeding. The aim of the research was to generate protocol of establishing Java cardamom micropropagation.  This research consisted of 4 aspects i.e. shoot induction of mother plant (without Plant Growth Regulator (PGR) and using PGR BAP (6-benzylaminopurine) 1000 ppm), explant origin selection (main stem, rhizome bud height >3 cm, rhizome bud height ≤3 cm and lateral rhizome), sterilization procedure establishment (4 methods differ in the use of  detergent, HgCl2, Alcohol, NaOCl, Ethanol, Iodine and soaking time in fungicide and bactericide) and shoot multiplication (MS 0, MS 0 + BAP 1 ppm and MS 0 + BAP 1 ppm + NAA 1 ppm). Result showed the application of 1000 ppm BAP to mature plant could induce shoot emergence.  The best explant source was rhizome bud that smaller or equal to 3 cm.  The highest survival rate (71%) was recorded when explants disinfected with 70% alcohol for 30 seconds and 0.1 % mercuric chloride for 5 minutes.  Java cardamom in vitro culture showed highest shoot multiplication rate in MS 0 + BAP 1 ppm medium (multiplier of 10 shoots/explant in 18 weeks).  Keywords: explant, in vitro propagation, Plant Growth Regulator, rhizome, shoot multiplicatio

Nitrate restricts nodule organogenesis through inhibition of cytokinin biosynthesis in Lotus japonicus

Legumes balance nitrogen acquisition from soil nitrate with symbiotic nitrogen fixation. Nitrogen fixation requires establishment of a new organ, which is a cytokinin dependent developmental process in the root. We found cytokinin biosynthesis is a central integrator, balancing nitrate signalling with symbiotic acquired nitrogen. Low nitrate conditions provide a permissive state for induction of cytokinin by symbiotic signalling and thus nodule development. In contrast, high nitrate is inhibitory to cytokinin accumulation and nodule establishment in the root zone susceptible to nodule formation. This reduction of symbiotic cytokinin accumulation was further exacerbated in cytokinin biosynthesis mutants, which display hypersensitivity to nitrate inhibition of nodule development, maturation and nitrogen fixation. Consistent with this, cytokinin application rescues nodulation and nitrogen fixation of biosynthesis mutants in a concentration dependent manner. These inhibitory impacts of nitrate on symbiosis occur in a Nlp1 and Nlp4 dependent manner and contrast with the positive influence of nitrate on cytokinin biosynthesis that occurs in species that do not form symbiotic root nodules. Altogether this shows that legumes, as exemplified by Lotus japonicus, have evolved a different cytokinin response to nitrate compared to non-legumes

Transforming, Genome Editing and Phenotyping the Nitrogen-fixing Tropical Cannabaceae Tree Parasponia andersonii

Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae). Together with 4 additional species, it forms the only known non-legume lineage able to establish a nitrogen-fixing nodule symbiosis with rhizobium. Comparative studies between legumes and P. andersonii could provide valuable insight into the genetic networks underlying root nodule formation. To facilitate comparative studies, we recently sequenced the P. andersonii genome and established Agrobacterium tumefaciens-mediated stable transformation and CRISPR/Cas9-based genome editing. Here, we provide a detailed description of the transformation and genome editing procedures developed for P. andersonii. In addition, we describe procedures for the seed germination and characterization of symbiotic phenotypes. Using this protocol, stable transgenic mutant lines can be generated in a period of 2-3 months. Vegetative in vitro propagation of T0 transgenic lines allows phenotyping experiments to be initiated at 4 months after A. tumefaciens co-cultivation. Therefore, this protocol takes only marginally longer than the transient Agrobacterium rhizogenes-based root transformation method available for P. andersonii, though offers several clear advantages. Together, the procedures described here permit P. andersonii to be used as a research model for studies aimed at understanding symbiotic associations as well as potentially other aspects of the biology of this tropical tree.</p

Duplication of symbiotic lysin motif receptors predates the evolution of nitrogen-fixing nodule symbiosis

Rhizobium nitrogen-fixing nodule symbiosis occurs in two taxonomic lineages: legumes (Fabaceae) and the genus Parasponia (Cannabaceae). Both symbioses are initiated upon the perception of rhizobium-secreted lipochitooligosaccharides (LCOs), called Nod factors. Studies in the model legumes Lotus japonicus and Medicago truncatula showed that rhizobium LCOs are perceived by a heteromeric receptor complex of distinct Lys motif (LysM)-type transmembrane receptors named NOD FACTOR RECEPTOR1 (LjNFR1) and LjNFR5 (L. japonicus) and LYSM DOMAIN CONTAINING RECEPTOR KINASE3 (MtLYK3)-NOD FACTOR PERCEPTION (MtNFP; M. truncatula). Recent phylogenomic comparative analyses indicated that the nodulation traits of legumes, Parasponia spp., as well as so-called actinorhizal plants that establish a symbiosis with diazotrophic Frankia spp. bacteria share an evolutionary origin about 110 million years ago. However, the evolutionary trajectory of LysM-type LCO receptors remains elusive. By conducting phylogenetic analysis, transcomplementation studies, and CRISPR-Cas9 mutagenesis in Parasponia andersonii, we obtained insight into the origin of LCO receptors essential for nodulation. We identified four LysM-type receptors controlling nodulation in P. andersonii: PanLYK1, PanLYK3, PanNFP1, and PanNFP2. These genes evolved from ancient duplication events predating and coinciding with the origin of nodulation. Phylogenetic and functional analyses associated the occurrence of a functional NFP2-orthologous receptor to LCO-driven nodulation. Legumes and Parasponia spp. use orthologous LysM-type receptors to perceive rhizobium LCOs, suggesting a shared evolutionary origin of LCO-driven nodulation. Furthermore, we found that both PanLYK1 and PanLYK3 are essential for intracellular arbuscule formation of mutualistic endomycorrhizal fungi. PanLYK3 also acts as a chitin oligomer receptor essential for innate immune signaling, demonstrating functional analogy to CHITIN ELECITOR RECEPTOR KINASE-type receptors.</p

Parallel loss of symbiosis genes in relatives of nitrogen-fixing non-legume Parasponia

Rhizobium nitrogen-fixing nodules are a well-known trait of legumes, but nodules also occur in other plant lineages either with rhizobium or the actinomycete Frankia as microsymbiont. The widely accepted hypothesis is that nodulation evolved independently multiple times, with only a few losses. However, insight in the evolutionary trajectory of nodulation is lacking. We conducted comparative studies using Parasponia (Cannabaceae), the only non-legume able to establish nitrogen fixing nodules with rhizobium. This revealed that Parasponia and legumes utilize a large set of orthologous symbiosis genes. Comparing genomes of Parasponia and its non-nodulating relative Trema did not reveal specific gene duplications that could explain a recent gain of nodulation in Parasponia. Rather, Trema and other non-nodulating species in the order Rosales show evidence of pseudogenization or loss of key symbiosis genes. This demonstrates that these species have lost the potential to nodulate. This finding challenges a long-standing hypothesis on evolution of nitrogen-fixing symbioses, and has profound implications for translational approaches aimed at engineering nitrogen-fixing nodules in crop plants

Trema orientale Genome sequencing and assembly

Genome and transcriptome sequencing of Trema orientalis RG33-

Comparative genomics of the nonlegume Parasponia reveals insights into evolution of nitrogen-fixing rhizobium symbioses

Nodules harboring nitrogen-fixing rhizobia are a well-known trait of legumes, but nodules also occur in other plant lineages, with rhizobia or the actinomycete Frankia as microsymbiont. It is generally assumed that nodulation evolved independently multiple times. However, molecular-genetic support for this hypothesis is lacking, as the genetic changes underlying nodule evolution remain elusive. We conducted genetic and comparative genomics studies by using Parasponia species (Cannabaceae), the only nonlegumes that can establish nitrogen-fixing nodules with rhizobium. Intergeneric crosses between Parasponia andersonii and its nonnodulating relative Trema tomentosa demonstrated that nodule organogenesis, but not intracellular infection, is a dominant genetic trait. Comparative transcriptomics of P. andersonii and the legume Medicago truncatula revealed utilization of at least 290 orthologous symbiosis genes in nodules. Among these are key genes that, in legumes, are essential for nodulation, including NODULE INCEPTION (NIN) and RHIZOBIUM-DIRECTED POLAR GROWTH (RPG). Comparative analysis of genomes from three Parasponia species and related nonnodulating plant species show evidence of parallel loss in nonnodulating species of putative orthologs of NIN, RPG, and NOD FACTOR PERCEPTION. Parallel loss of these symbiosis genes indicates that these nonnodulating lineages lost the potential to nodulate. Taken together, our results challenge the view that nodulation evolved in parallel and raises the possibility that nodulation originated ∼100 Mya in a common ancestor of all nodulating plant species, but was subsequently lost in many descendant lineages. This will have profound implications for translational approaches aimed at engineering nitrogen-fixing nodules in crop plants