Meta-analysis of biodynamic (BD) preparations reveal the bacterial population involved in improving soil health, crop yield and quality.

BACKGROUND: Bacterial community found in biodynamic preparations (BD500-BD507) can help improve soil health, plant development, yield, and quality. The current work describes a metagenomic investigation of these preparations to identify the bacterial communities along with the functional diversity present within them. RESULTS: Metagenome sequencing was performed using the Illumina MiSeq platform, which employs next-generation sequencing (NGS) technology, to provide an understanding of the bacterial communities and their functional diversity in BD preparations. NGS data of BD preparations revealed that maximum operational taxonomic units (OTUs) of the phylum Proteobacteria were present in BD506 (23429) followed by BD505 (22712) and BD501 (21591), respectively. Moreover, unclassified phylum (16657) and genus (16657) were also highest in BD506. Maximum alpha diversity was reported in BD501 (1095 OTU) and minimum in BD507 (257 OTU). Further, the OTUs for five major metabolic functional groups viz carbohydrate metabolism, xenobiotic degradation, membrane transport functions, energy metabolism, and enzyme activities were abundant in BD506 and BD501. CONCLUSION: The bacterial communities in BD506 and BD501 are found to be unique and rare; they belong to functional categories that are involved in enzyme activity, membrane transport, xenobiotic degradation, and carbohydrate metabolism. These preparations might therefore be thought to be more effective. The investigation also found a highly varied population of bacteria, which could explain why BD preparations work well in the field. In view of this, the BD preparations may be utilized for unexploited bacterial communities for sustainable agriculture production.

Immobilization of microbial multienzyme preparation on calcium alginate beads as well as lyophilization with mosambi peel matrix improved its shelf-life and stability.

The purpose of the current study was to evaluate the functional activity and storage viability (at 4 °C and 35 °C) of an immobilized as well as lyophilized multienzyme, viz., pectinase, cellulase, and amylase (PCA) that was produced by Bacillus subtilis NG105 under solid state fermentation (SSF) at 35 ℃ for 10 days using mosambi peel as a substrate. After SSF, the culture media was divided into two aliquots. From the first aliquot, the produced ME was extracted, precipitated, and further immobilized on calcium alginate beads (MEICA). In order to immobilize on mosambi peel matrix, the second aliquot was mixed with acetone and subsequently lyophilized (MELMP). Thus, ready MEICA and MELMP extracted 87.5 and 91.5% juice from mango pulp, respectively. In the reusability study, after 5 cycles, MEICA exhibited 23.8%, 24.4%, and 36.5% PCA activity, respectively. The PCA activity of MEICA and MELMP was examined after 60 days of storage at 4 ℃. The result revealed that the PCA for MEICA declined from 100 to 66%, 58.2%, and 64.5%, respectively, while for MELMP, it dropped from 100 to 84.2%, 82.1%, and 69.7%, respectively. Further, after 60 days of storage, the reduction of total protein content (TPC) in free multienzyme (FME), MEICA, and MELMP was 92.2%, 91.5%, and 36.3% observed, respectively. In the localization study, the maximum levels of multienzyme activity were found in cell exudates. This study demonstrated that immobilizing of multienzyme through lyophilization on waste substrates like mosambi peel boosted its stability and shelf-life along with greatly reducing the cost of products.

In-silico mining and characterization of MYB family genes in wilt-resistant hybrid guava (Psidium guajava × Psidium molle).

BACKGROUND: The MYB family is one of the most significant groups of transcription factors in plants. However, several MYBs have been linked to secondary metabolism and are important for determining the color of fruit's peel and pulp. Despite being a substantial fruit crop in tropical and subtropical areas of the world, wilt-resistant hybrid guava (Psidium guajava × Psidium molle; PGPM) has not yet been the subject of a thorough examination. This study's goal was to assess the expression of MYB in guava fruit pulp, roots, and seeds to predict its function by in silico analysis of the guava root transcriptome data. RESULTS: In the current study, we have mined the MYBs family of MYB genes from the transcriptome of the PGPM guava root. We have mined 15 distinct MYB transcription factor genes/transcripts viz MYB3, MYB4, MYB23, MYB86, MYB90, MYB308, MYB5, MYB82, MYB114, MYB6, MYB305, MYB44, MYB51, MYB46, and MYB330. From the analyses, it was found that R2-MYB and R3-MYB domains are conserved in all known guava MYB proteins. The expression of six different MYB TFs was examined using semi-quantitative RT-PCR in "Shweta" pulp (white colour pulp), "Lalit" pulp (red color pulp), "Lalit" root, and "Lalit" seed. CONCLUSION: There were 15 MYB family members observed in guava. They were unequally distributed across the chromosomes, most likely as a result of gene duplication. Additionally, the expression patterns of the particular MYBs showed that MYB may be involved in the control of wilt, fruit ripening, seed development, and root development. Our results allow for a more thorough functional characterization of the guava MYB family genes and open the door to additional research into one essential MYB transcription factor family of genes and its involvement in the growth and ripening of guava fruit.

Hexavalent chromium-reducing plant growth-promoting rhizobacteria are utilized to bio-fortify trivalent chromium in fenugreek by promoting plant development and decreasing the toxicity of hexavalent chromium in the soil.

BACKGROUND: Fenugreek is known to have good anti-diabetes properties. Moreover, several studies accounted that the trivalent form of chromium [Cr(III)] also have anti-diabetic properties. However, its hexavalent form i.e., Cr(VI) is known to be highly toxic and carcinogenic to living beings and retarded plant growth even if it is present in low concentration in soil. Many plant growth-promoting rhizobacteria (PGPR) are reported to have the potential to reduce the Cr(VI) into Cr(III) in soil. In view of the above, the present objective was designed to effectively utilize Cr(VI) reducing PGPRs for the growth and development of fenugreek plant in Cr(VI) amended soil, apart from reducing Cr(VI) in soil and fortification of Cr(III) in the aerial part of plants. METHODS: The experiment was carried out to evaluate the effect of Cr(VI)-reducing PGPRs viz. Bacillus cereus (SUCR44); Microbacterium sp. (SUCR140); Bacillus thuringiensis (SUCR186) and B. subtilis (SUCR188) on growth, uptake and translocation of Cr as well as other physiological parameters in fenugreek grown under artificially Cr(VI) amended soil (100 mg kg-1 of Cr(VI) in soil). RESULTS: The aforementioned concentration of Cr(VI) in soil cause severe reduction in root length (41 %), plant height (43 %), dry root (38 %) and herb biomass (48 %), when compared with control negative (CN; uninoculated plant not grown in Cr(VI) contaminated soil). However, the presence of Microbacterium sp.-SURC140 (MB) mitigates the Cr toxicity resulting in improved root length (92 %), plant height (86 %), dry root (74 %) and herb biomass (99 %) as compared with control positive (CP; uninoculated plants grown in Cr(VI) contaminated soil). The maximum reduction in bioavailability (82 %) of Cr(VI) in soil and its uptake (50 %) by the plant were also observed in MB-treated plants. However, All Cr(VI)-reducing PGPRs failed to decrease the translocation of Cr to the aerial parts. Moreover, the plant treated with MB observed diminution in relative water content (13 %), electrolyte leakage (16%) and lipid peroxidation (38 %) as well as higher chlorophyll (37 %) carotenoids (17 %) contents and antioxidants (18%) potential. CONCLUSION: This study demonstrates that MB can lower the Cr(VI) toxicity to the plant by reducing the bioavailable Cr(VI), consequently reducing the Cr(VI) toxicity level in soil and helping in improving the growth and yield of fenugreek. Additionally, Cr(III) uptakes and translocation may improve the effectiveness of fenugreek in treating diabetes.

Management of chromium(VI)-contaminated soils through synergistic application of vermicompost, chromate reducing rhizobacteria and Arbuscular mycorrhizal fungi (AMF) reduced plant toxicity and improved yield attributes in Ocimum basilicum L.

An integrated approach involving vermicompost, chromate reducing bacteria and AMF was tested to manage the toxic impacts of Cr(VI) on Ocimum basilicum as a model plant. Pot experiments were conducted on O. basilicum plants in an artificially Cr(VI)-contaminated soil in two phases of experiment as bioinoculants experiment and vermicompost experiment. In the first phase of the bioinoculants experiment the series of gradient concentrations of Cr(VI) (0, 25, 50 and 100 mg kg-1 in soil) were evaluated with previously isolated four efficient Cr(VI)-reducing rhizo-bacterial strains (Bacillus Cereus strain SUCR 44, BC; Microbacterium sp. strain SUCR 140, MB; Bacillus thuringiensis strain SUCR186, BT; and Bacillus subtilis strain SUCR188; BS) along with Arbuscular Mycorrhizal Fungus-Glomus fasciculatum (GF) in alone and in co-inoculation form. In the second experiment (vermicompost) the best performing strain (MB) was tested alone or in combination with GF along with different doses of vermicompost. It was observed that vermicompost by itself could be useful in decreasing the bioavailable Cr(VI), uptake of Cr besides improving the nutritional status of plants. The vermicompost also played an important and indirect role and improved herb yield by supporting the multiplication of MB (Microbacterium sp.), an efficient chromate reducing rhizobacteria, that further decreased the bioavailable and toxic form of Cr and improved population and colonization of GF too. The translocation of Cr(VI) was averted through improved colonization of GF, also prevented higher accumulation of Cr in aerial parts (leafy herb) of O. basilicum.

Mechanism and kinetics of chlorpyrifos co-metabolism by using environment restoring microbes isolated from rhizosphere of horticultural crops under subtropics.

The indiscriminate use of organophosphate insecticide chlorpyrifos in agricultural crops causes significant soil and water pollution and poses a serious threat to the global community. In this study, a microbial consortium ERM C-1 containing bacterial strains Pseudomonas putida T7, Pseudomonas aeruginosa M2, Klebsiella pneumoniae M6, and a fungal strain Aspergillus terreus TF1 was developed for the effective degradation of chlorpyrifos. Results revealed that microbial strains were not only utilizing chlorpyrifos (500 mg L-1) but also coupled with plant growth-promoting characteristics and laccase production. PGP traits, that is, IAA (35.53, 45.53, 25.19, and 25.53 µg mL-1), HCN (19.85, 17.85, 12.18, and 9.85 µg mL-1), and ammonium (14.73, 16.73, 8.05, and 10.87 µg mL-1) production, and potassium (49.53, 66.72, 46.14, and 52.72 µg mL-1), phosphate (52.37, 63.89, 33.33, and 71.89 µg mL-1), and zinc (29.75, 49.75, 49.12, and 57.75 µg mL-1) solubilization tests were positive for microbial strains T7, M2, M6, and TF1, respectively. The laccase activity by ERM C-1 was estimated as 37.53, 57.16, and 87.57 enzyme U mL-1 after 5, 10, and 15 days of incubation, respectively. Chlorpyrifos degradation was associated with ERM C-1 and laccase activity, and the degree of enzyme activity was higher in the consortium than in individual strains. The biodegradation study with developed consortium ERM C-1 showed a decreased chlorpyrifos concentration from the 7th day of incubation (65.77% degradation) followed by complete disappearance (100% degradation) after the 30th day of incubation in the MS medium. First-order degradation kinetics with a linear model revealed a high k -day value and low t 1/2 value in ERM C-1. The results of HPLC and GC-MS analysis proved that consortium ERM C-1 was capable of completely removing chlorpyrifos by co-metabolism mechanism.

Microbial production of multienzyme preparation from mosambi peel using Trichoderma asperellum.

Fruit and vegetable wastes create unhygienic conditions and pose a environmental pollution. The utilization of such wastes as carbon sources for production of enzyme with microbial intervention could be an ecofriendly and profitable approach, apart from diminishing the waste load. The present investigation focused on the feasibility of using mosambi (Citrus limetta) peel as substrate for multienzyme production (pectinase, cellulase and amylase) through microbial intervention. Fifteen fungi were isolated from organic waste and screened in vitro their potential of biodegradation of mosambi peel through enzymes production. The best performing isolate was selected and identified as Trichoderma asperellum NG-125 (accession number-MW287256). Conditions viz. temperature, pH, incubation time and nutrient addition were optimized for efficient enzymes production. The maximum enzyme activity (U ml-1 min-1) of pectinase (595.7 ± 2.47), cellulase (497.3 ± 2.06) and amylase (440.9 ± 1.44) were observed at pH 5.5, incubation temperature of 30 °C after 10 days of fermentation. Moreover, macro-nutrients such as ammonium sulfate (0.1%) and potassium-di-hydrogen-ortho-phosphate (0.01%) further also enhanced the production of enzymes. The SDS-PAGE analysis of purified pectinase, cellulase and amylase using showed molecular mass of 43, 66 and 33 kDa, respectively. The enzyme retention activity (ERA) of aforesaid enzymes was also tested with four different natural fiber matrices viz., bagasse, rice husk, paddy straw and wheat straw. Among these, the maximum ERA was observed on bagasse matrix (pectinase-56.35%, cellulose-77.68% and amylase 59.54%). Enzymatic juice clarification yield obtained with test enzyme was 75.8%, as compared to 80.5% of commercial enzyme. The result indicates that T. asperellum may be exploited as multifaceted biocatalysis.

Papaya Leaf Curl Virus (PaLCuV) Infection on Papaya (Carica papaya L.) Plants Alters Anatomical and Physiological Properties and Reduces Bioactive Components.

Papaya leaves are used frequently for curing scores of ailments. The medicinal properties of papaya leaves are due to presence of certain bioactive/pharmacological compounds. However, the papaya leaf curl virus (PaLCuV), a geminivirus, is a major threat to papaya cultivation globally. During the present investigation, we observed that PaLCuV infection significantly altered the anatomy, physiology, and bioactive properties of papaya leaves. As compared to healthy leaves, the PaLCuV-infected leaves were found to have reduced stomatal density (76.83%), stomatal conductance (78.34%), photosynthesis rate (74.87%), water use efficiency (82.51%), chlorophyll (72.88%), carotenoid (46.63%), osmolality (48.55%), and soluble sugars (70.37%). We also found lower enzymatic activity (superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT)-56.88%, 85.27%, and 74.49%, respectively). It was found that the size of guard cells (50%), transpiration rate (45.05%), intercellular CO2 concentration (47.81%), anthocyanin (27.47%), proline content (74.17%), malondialdehyde (MDA) (106.65%), and electrolyte leakage (75.38%) was elevated in PaLCuV-infected leaves. The chlorophyll fluorescence analysis showed that the infected plant leaves had a significantly lower value of maximal quantum yield of photosystem II (PSII (Fv/Fm), photochemical quantum yield of photosystem I (PSI (Y(I)), and effective quantum yield of PSII (Y(II)). However, in non-photochemical quenching mechanisms, the proportion of energy dissipated in heat form (Y(NPQ)) was found to be significantly higher. We also tested the bioactivity of infected and healthy papaya leaf extracts on a Caenorhabditis elegans (C. elegans) model system. It was found that the crude extract of papaya leaves significantly enhanced the life span of C. elegans (29.7%) in comparison to virus-infected leaves (18.4%) on application of 100 µg/mL dose of the crude extract. Our research indicates that the PaLCuV-infected leaves not only had anatomical and physiological losses, but that pharmacological potential was also significantly decreased.

Isolation and characterization of endophytic fungi having plant growth promotion traits that biosynthesizes bacosides and withanolides under in vitro conditions.

Endophytes are regarded with immense potentials in terms of plant growth promoting (PGP) elicitors and mimicking secondary metabolites of medicinal importance. Here in the present study, we explored Bacopa monnieri plants to isolate, identify fungal endophytes with PGP elicitation potentials, and investigate secretion of secondary metabolites such as bacoside and withanolide content under in vitro conditions. Three fungal endophytes isolated (out of 40 saponin producing isolates) from leaves of B. monnieri were examined for in vitro biosynthesis of bacosides. On morphological, biochemical, and molecular identification (ITS gene sequencing), the isolated strains SUBL33, SUBL51, and SUBL206 were identified as Nigrospora oryzae (MH071153), Alternaria alternata (MH071155), and Aspergillus terreus (MH071154) respectively. Among these strains, SUBL33 produced highest quantity of Bacoside A3 (4093 µg mL-1), Jujubogenin isomer of Bacopasaponin C (65,339 µg mL-1), and Bacopasaponin C (1325 µg mL-1) while Bacopaside II (13,030 µg mL-1) was produced by SUBL51 maximally. Moreover, these aforementioned strains also produced detectable concentration of withanolides-Withaferrin A, Withanolide A (480 µg mL-1), and Withanolide B (1024 µg mL-1) respectively. However, Withanolide A was not detected in the secondary metabolites of strain SUBL51. To best of our knowledge, the present study is first reports of Nigrospora oryzae as an endophyte in B. monnieri with potentials of biosynthesis of economically important phytomolecules under in vitro conditions.

Complementarity among plant growth promoting traits in rhizospheric bacterial communities promotes plant growth.

An assessment of roles of rhizospheric microbial diversity in plant growth is helpful in understanding plant-microbe interactions. Using random combinations of rhizospheric bacterial species at different richness levels, we analysed the contribution of species richness, compositions, interactions and identity on soil microbial respiration and plant biomass. We showed that bacterial inoculation in plant rhizosphere enhanced microbial respiration and plant biomass with complementary relationships among bacterial species. Plant growth was found to increase linearly with inoculation of rhizospheric bacterial communities with increasing levels of species or plant growth promoting trait diversity. However, inoculation of diverse bacterial communities having single plant growth promoting trait, i.e., nitrogen fixation could not enhance plant growth over inoculation of single bacteria. Our results indicate that bacterial diversity in rhizosphere affect ecosystem functioning through complementary relationship among plant growth promoting traits and may play significant roles in delivering microbial services to plants.

Biodiversity acts as insurance of productivity of bacterial communities under abiotic perturbations.

Anthropogenic disturbances are detrimental to the functioning and stability of natural ecosystems. Critical ecosystem processes driven by microbial communities are subjected to these disturbances. Here, we examine the stabilizing role of bacterial diversity on community biomass in the presence of abiotic perturbations such as addition of heavy metals, NaCl and warming. Bacterial communities with a diversity gradient of 1-12 species were subjected to the different treatments, and community biomass (OD600) was measured after 24 h. We found that initial species richness and phylogenetic structure impact the biomass of communities. Under abiotic perturbations, the presence of tolerant species in community largely contributed in community biomass production. Bacterial diversity stabilized the biomass across the treatments, and differential response of bacterial species to different perturbations was the key reason behind these effects. The results suggest that biodiversity is crucial for maintaining the stability of ecosystem functioning and acts as ecological insurance under abiotic perturbations. Biodiversity in natural ecosystems may also uphold the ecosystem functioning under anthropogenic disturbance.

Pretreatment of Cr(VI)-amended soil with chromate-reducing rhizobacteria decreases plant toxicity and increases the yield of Pisum sativum.

Pot culture experiments were performed under controlled greenhouse conditions to investigate whether four Cr(VI)-reducing bacterial strains (SUCR44, SUCR140, SUCR186, and SUCR188) were able to decrease Cr toxicity to Pisum sativum plants in artificially Cr(VI)-contaminated soil. The effect of pretreatment of soil with chromate-reducing bacteria on plant growth, chromate uptake, bioaccumulation, nodulation, and population of Rhizobium was found to be directly influenced by the time interval between bacterial treatment and seed sowing. Pretreatment of soil with SUCR140 (Microbacterium sp.) 15 days before sowing (T+15) showed a maximum increase in growth and biomass in terms of root length (93 %), plant height (94 %), dry root biomass (99 %), and dry shoot biomass (99 %). Coinoculation of Rhizobium with SUCR140 further improved the aforementioned parameter. Compared with the control, coinoculation of SUCR140+R showed a 117, 116, 136, and 128 % increase, respectively, in root length, plant height, dry root biomass, and dry shoot biomass. The bioavailability of Cr(VI) decreased significantly in soil (61 %) and in uptake (36 %) in SUCR140-treated plants; the effects of Rhizobium, however, either alone or in the presence of SUCR140, were not significant. The populations of Rhizobium (126 %) in soil and nodulation (146 %) in P. sativum improved in the presence of SUCR140 resulting in greater nitrogen (54 %) concentration in the plants. This study shows the usefulness of efficient Cr(VI)-reducing bacterial strain SUCR140 in improving yields probably through decreased Cr toxicity and improved symbiotic relationship of the plants with Rhizobium. Further decrease in the translocation of Cr(VI) through improved nodulation by Rhizobium in the presence of efficient Cr-reducing bacterial strains could also decrease the accumulation of Cr in shoots.

A Cr(VI)-reducing Microbacterium sp. strain SUCR140 enhances growth and yield of Zea mays in Cr(VI) amended soil through reduced chromium toxicity and improves colonization of arbuscular mycorrhizal fungi.

Pot culture experiments were conducted in a glasshouse to evaluate the effects of four efficient Cr(VI)-reducing bacterial strains (SUCR44, SUCR140, SUCR186, and SUCR188) isolated from rhizospheric soil, and four arbuscular mycorrhizal fungi (AMF-Glomus mosseae, G. aggregatum, G. fasciculatum, and G. intraradices) alone or in combination, on Zea mays in artificially Cr(VI)-amended soil. Presence of a strain of Microbacterium sp. SUCR140 reduced the chromate toxicity resulting in improved growth and yields of plants compared to control. The bioavailability of Cr(VI) in soil and its uptake by the plant reduced significantly in SUCR140-treated plants; the effects of AMF, however, either alone or in presence of SUCR140 were not significant. On the other hand, presence of AMF significantly restricted the transport of chromium from root to the aerial parts of plants. The populations of AMF chlamydospores in soil and its root colonization improved in presence of SUCR140. This study demonstrates the usefulness of an efficient Cr(VI)-reducing bacterial strain SUCR140 in improving yields probably through reducing toxicity to plants by lowering bioavailability and uptake of Cr(VI) and improving nutrient availability through increased mycorrhizal colonization which also restricted the transport of chromium to the aerial parts.

In vitro Cr(VI) reduction by cell-free extracts of chromate-reducing bacteria isolated from tannery effluent irrigated soil.

Four efficient Cr(VI)-reducing bacterial strains were isolated from rhizospheric soil of plants irrigated with tannery effluent and investigated for in vitro Cr(VI) reduction. Based on 16S rRNA gene sequencing, the isolated strains SUCR44, SUCR140, SUCR186, and SUCR188 were identified as Bacillus sp. (JN674188), Microbacterium sp. (JN674183), Bacillus thuringiensis (JN674184), and Bacillus subtilis (JN674195), respectively. All four isolates could completely reduce Cr(VI) in culture media at 0.2 mM concentration within a period of 24-120 h; SUCR140 completely reduced Cr(VI) within 24 h. Assay with the permeabilized cells (treated with Triton X-100 and Tween 80) and cell-free assay demonstrated that the Cr(VI) reduction activity was mainly associated with the soluble fraction of cells. Considering the major amount of chromium being reduced within 24-48 h, these fractions could have been released extracellularly also during their growth. At the temperature optima of 28 °C and pH 7.0, the specific activity of Cr(VI) reduction was determined to be 0.32, 0.42, 0.34, and 0.28 µmol Cr(VI)min(-1)mg(-1) protein for isolates SUCR44, SUCR140, SUCR186, and SUCR188, respectively. Addition of 0.1 mM NADH enhanced the Cr(VI) reduction in the cell-free extracts of all four strains. The Cr(VI) reduction activity in cell-free extracts of all the isolates was stable in presence of different metal ions tested except Hg(2+). Beside this, urea and thiourea also reduced the activity of chromate reduction to significant levels.

Synergy between Glomus fasciculatum and a beneficial Pseudomonas in reducing root diseases and improving yield and forskolin content in Coleus forskohlii Briq. under organic field conditions.

Root rot and wilt, caused by a complex involving Fusarium chlamydosporum (Frag. and Cif.) and Ralstonia solanacearum (Smith), are serious diseases affecting the cultivation of Coleus forskohlii, a crop with economic potential as a source of the medicinal compound forskolin. The present 2-year field experiments were conducted with two bioinoculants (a native Pseudomonas monteilii strain and the exotic arbuscular mycorrhizal (AM) fungus Glomus fasciculatum) alone and in combination under organic field conditions in order to evaluate their potential in controlling root rot and wilt. Combined inoculation of P. monteilii with G. fasciculatum significantly increased plant height, plant spread, and number of branches; reduced disease incidence; and increased tuber dry mass of C. forskohlii, compared to vermicompost controls not receiving any bioinoculants. Increase in tuber yields was accompanied by an increase in plant N, P, and K uptake. Co-inoculation of P. monteilii with G. fasciculatum significantly improved the percent AM root colonization and spore numbers retrieved from soil. This suggests P. monteilii to be a mycorrhiza helper bacterium which could be useful in organic agriculture. The forskolin content of tubers was significantly increased by the inoculation treatments of P. monteilii, G. fasciculatum, and P. monteilii + G. fasciculatum.