Diversity of Native Arbuscular Mycorrhiza Extraradical Mycelium Influences Antioxidant Enzyme Activity in Wheat Grown Under Mn Toxicity.

Sustainable agricultural practices based on the development of native arbuscular mycorrhizal fungi (AMF) can improve crop growth and stress tolerance in acidic soils with manganese toxicity. The beneficial effects are stronger when crops are colonized early in development by an intact extraradical mycelium (ERM), but are dependent on AMF assemblage. In wheat colonized by AMF associated to Lolium rigidum L. (LOL) or Ornithopus compressus (ORN), growth and stress tolerance are differently influenced. In the present study, this functional diversity was studied by evaluating the activity of ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR), guaiacol peroxidase (GPX), superoxide dismutase (SOD) and Mn-SOD. ORN treatment promoted higher wheat shoot and root dry weights, a higher root protein content, decreased root APX, GR and SOD activities but a higher proportion of MnSOD activity. ORN associated microbiota differently manage antioxidant enzyme activity of succeeding wheat to improve growth.

Toxic levels of manganese in an acidic Cambisol alters antioxidant enzymes activity, element uptake and subcellular distribution in Triticum aestivum.

In the Montado system, in Portuguese Alentejo region, some Eutric Cambisols are known to promote manganese (Mn) toxicity in wheat. Variation on bioavailable Mn concentration depends on soil acidity, which can be increased by natural events (e.g. waterlogging) or human activity (e.g. excess use of chemical fertilizers). The effect of increasing soil Mn on crop element uptake, element distribution and oxidative stress was evaluated on winter wheat (Triticum aestivum). Plants were grown for 3 weeks in an acidic Cambisol spiked with increasing Mn concentrations (0, 45.2 and 90.4 mg MnCl2/Kg soil). Calcium (Ca), phosphorus (P), magnesium (Mg) and Mn were quantified in the soil solution, root and shoot tissues and respective subcellular fractions. The activity of the antioxidant enzymes ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR), guaiacol peroxidase (GPX) and superoxide dismutase (SOD) were determined in extracts of wheat shoots and roots. Overall, increase in soil bioavailable Mn inhibited the uptake of other elements, increased the Ca proportion in the root apoplast, promoted the translocation of Mn and P to shoot tissues and increased their proportion in the shoot vacuoles. Wheat roots showed greater antioxidant enzymes activities than shoots. These activities decreased at the highest soil Mn concentration in both plant parts. Wheat roots appear to be more sensitive to oxidative stress derived from excess soil Mn and promote Mn translocation and storage in shoot vacuoles, probably in Mn and P complexes, as a detoxification strategy. Improvement in wheat production, in acidic soils, may rely on the enhancement of its Mn detoxification strategies.

In vitro co-cultures of Pinus pinaster with Bursaphelenchus xylophilus: a biotechnological approach to study pine wilt disease.

MAIN CONCLUSION: Co-cultures of Pinus pinaster with Bursaphelenchus xylophilus were established as a biotechnological tool to evaluate the effect of nematotoxics addition in a host/parasite culture system. The pinewood nematode (PWN), Bursaphelenchus xylophilus, the causal agent of pine wilt disease (PWD), was detected for the first time in Europe in 1999 spreading throughout the pine forests in Portugal and recently in Spain. Plant in vitro cultures may be a useful experimental system to investigate the plant/nematode relationships in loco, thus avoiding the difficulties of field assays. In this study, Pinus pinaster in vitro cultures were established and compared to in vivo 1 year-old plantlets by analyzing shoot structure and volatiles production. In vitro co-cultures were established with the PWN and the effect of the phytoparasite on in vitro shoot structure, water content and volatiles production was evaluated. In vitro shoots showed similar structure and volatiles production to in vivo maritime pine plantlets. The first macroscopic symptoms of PWD were observed about 4 weeks after in vitro co-culture establishment. Nematode population in the culture medium increased and PWNs were detected in gaps of the callus tissue and in cavities developed from the degradation of cambial cells. In terms of volatiles main components, plantlets, P. pinaster cultures, and P. pinaster with B. xylophilus co-cultures were all ß- and α-pinene rich. Co-cultures may be an easy-to-handle biotechnological approach to study this pathology, envisioning the understanding of and finding ways to restrain this highly devastating nematode.

Phytochemical Volatiles as Potential Bionematicides with Safer Ecotoxicological Properties.

The management of plant-parasitic nematodes (PPNs) still relies on traditional nematicides that threaten the environment and human health. Novel solutions are urgently needed for PPN pest management that are effective while safeguarding non-target organisms. Volatile phytochemicals belong to a structurally diverse group of bioactive metabolites that are believed to hold safer environmental characteristics than synthetic pesticides. Nonetheless, not many studies have analysed the potential environmental benefits of shifting to these novel bionematicides. In the present study, 20 phytochemical volatiles with reported nematicidal activity were compared to traditional pesticides using specific parameters of environmental and human health safety available on applied online databases and predicted in silico through specialised software. Overall, the reviewed nematicidal phytochemicals were reportedly less toxic than synthetic nematicides. They were predicted to disperse to the air and soil environmental compartments and were reported to have a lower toxicity on aquatic organisms. On the contrary, the synthetic nematicides were reportedly toxic to aquatic organisms while showing a predicted high affinity to the water environmental compartment. As alternatives, ß-keto or fatty acid derivatives, e.g., aliphatic alcohols or ketones, showed more adequate properties. This study highlights the importance of complementing studies on nematicidal activity with a risk assessment-based analysis to allow for a faster selection of nematicidal phytochemical volatiles and to leverage the development and implementation of bionematicides.

Cymbopogon citratus Allelochemical Volatiles as Potential Biopesticides against the Pinewood Nematode.

Traditional pesticides are based on toxic compounds that can reduce biodiversity, degrade the environment, and contribute to less healthy living. Plant allelochemicals can provide more environmentally friendly and sustainable alternatives. Essential oils (EOs) are complex mixtures of plant secondary metabolites that show strong biological activities. In the present study, the EOs of Cymbopogon citratus were screened for activity against the pinewood nematode (PWN), the causal agent of pine wilt disease. To understand their nematicidal properties, EOs were fractioned into hydrocarbon molecules and oxygen-containing compounds, and their main compounds were acquired and tested separately against the PWN. The EO oxygen-containing molecules fraction was highly active against the PWN (EC50 = 0.279 µL/mL), with citral and geraniol showing higher activities (EC50 = 0.266 and 0.341 µL/mL, respectively) than emamectin benzoate (EC50 = 0.364 µL/mL), a traditional nematicide used against the PWN. These compounds were additionally reported to be less toxic to non-target organisms (fish, invertebrates, and algae) and safer to human health (with higher reported toxicity thresholds) and predicted to exert fewer environmental impacts than traditional nematicides. Resorting to approved natural compounds can quickly leverage the development of sustainable alternatives to traditional nematicides.

Nematicidal Activity of Volatiles against the Rice Root-Knot Nematode and Environmental Safety in Comparison to Traditional Nematicides.

The rice root-knot nematode (RRKN), Meloidogyne graminicola Golden and Birchfield 1965, is a dangerous crop pest that affects rice production on a global scale. The largest rice-producing countries struggle with the impacts of RRKN infestation, namely, underdeveloped plants and a reduction in rice grain that can reach up to 70% of crop yield. In addition, the shift to strategies of sustainable pest management is leading to a withdrawal of some of the most effective pesticides, given the dangers they pose to the environment and human health. Volatile metabolites produced by plants can offer safer alternatives. The present study characterized the nematicidal activity of volatile phytochemicals against the RRKN and compared the most active with commercial nematicides concerning their safety to the environment and human health. Rice plants were used to grow large numbers of RRKNs for direct-contact bioassays. Mortality induced by the volatiles was followed for four days on RRKN second-stage juveniles. Of the 18 volatiles tested, carvacrol, eugenol, geraniol, and methyl salicylate showed the highest mortalities (100%) and were compared to traditional nematicides using (eco)toxicological parameters reported on freely available databases. While methyl salicylate had a faster activity, carvacrol had more lasting effects. When compared to synthetic nematicides, these volatile phytochemicals were reported to have higher thresholds of toxicity and beneficial ecotoxicological parameters. Ultimately, finding safer alternatives to traditional pesticides can lower the use of damaging chemicals in farming and leverage the transition to agricultural practices with a lower impact on biodiversity.

Infection of In Vivo and In Vitro Pines with the Pinewood Nematode Bursaphelenchus xylophilus and Isolation of Induced Volatiles.

The pinewood nematode (PWN) is a phytoparasite that causes pine wilt disease (PWD) in conifer species. This plant parasitic nematode has heavily contributed to pine deforestation in Asian countries, e.g., Japan, China, and Korea. Over the last two decades, in Europe, Portugal and Spain have been greatly affected. Research on the mechanisms of PWN infection and/or PWD progression in susceptible host species relies on the controlled infection of pine seedlings under greenhouse conditions. This technique is laborious and mobilizes substantial economic and human resources. Additionally, it can be prone to variability that results from the genetic diversity associated with some pine species but also from the interference of external factors. As an alternative, in vitro co-cultures of pine with PWNs offer a more advantageous system for studying biochemical changes since they a) allow controlling single environmental or nutritional variables, b) occupy less space, c) require less time to obtain, and d) are free from contamination or from host genetic variation. The following protocol details the standard in vivo PWN infection of Pinus pinaster, the maritime pine, and the establishment of the novel in vitro co-cultures of pine shoots with the PWN as an improved methodology to study this phytoparasite influence on pine volatiles. PWN-induced volatiles are extracted from in vivo and in vitro infected pines by hydrodistillation and distillation-extraction, and the emitted volatiles are captured by solid phase microextraction (SPME), using fiber or packed column techniques.

Nematicidal Activity of Phytochemicals against the Root-Lesion Nematode Pratylenchus penetrans.

Plant-parasitic nematodes (PPNs) are highly damaging pests responsible for heavy losses in worldwide productivity in a significant number of important plant crops. Common pest management strategies rely on the use of synthetic chemical nematicides, which have led to serious concerns regarding their impacts on human health and the environment. Plant natural products, or phytochemicals, can provide a good source of agents for sustainable control of PPNs, due to their intrinsic characteristics such as higher biodegradability, generally low toxicity for mammals, and lower bioaccumulation in the environment. In this work, the nematicidal activity of 39 phytochemicals was determined against the root-lesion nematode (RLN) Pratylenchus penetrans using standard direct and indirect contact methodologies. Overall, the RLN was tolerant to the tested phytochemicals at the highest concentration, 2 mg/mL, seldom reaching full mortality. However, high activities were obtained for benzaldehyde, carvacrol, 3-octanol, and thymol, in comparison to other phytochemicals or the synthetic nematicide oxamyl. These phytochemicals were seen to damage nematode internal tissues but not its cuticle shape. Also, the environmental and (eco)toxicological parameters reported for these compounds suggest lower toxicity and higher safety of use than oxamyl. These compounds appear to be good candidates for the development of biopesticides for a more sustainable pest management strategy.

Mycorrhizal Colonization of Wheat by Intact Extraradical Mycelium of Mn-Tolerant Native Plants Induces Different Biochemical Mechanisms of Protection.

Soil with excess Mn induces toxicity and impairs crop growth. However, with the development in the soil of an intact extraradical mycelia (ERM) from arbuscular mycorrhizal fungi (AMF) symbiotic to native Mn-tolerant plants, wheat growth is promoted due to a stronger AMF colonization and subsequent increased protection against Mn toxicity. To determine the biochemical mechanisms of protection induced by this native ERM under Mn toxicity, wheat grown in soil from previously developed Lolium rigidum (LOL) or Ornithopus compressus (ORN), both strongly mycotrophic plants, was compared to wheat grown in soil from previously developed Silene gallica (SIL), a non-mycotrophic plant. Wheat grown after LOL or ORN had 60% higher dry weight, ca. two-fold lower Mn levels and almost double P contents. Mn in the shoots was preferentially translocated to the apoplast along with Mg and P. The activity of catalase increased; however, guaiacol peroxidase (GPX) and superoxide dismutase (SOD) showed lower activities. Wheat grown after ORN differed from that grown after LOL by displaying slightly higher Mn levels, higher root Mg and Ca levels and higher GPX and Mn-SOD activities. The AMF consortia established from these native plants can promote distinct biochemical mechanisms for protecting wheat against Mn toxicity.

First Report on the Synergistic Interaction between Essential Oils against the Pinewood Nematode Bursaphelenchus xylophilus.

Control of the pinewood nematode (PWN), the causal agent of pine wilt disease, can be achieved through the trunk injection of nematicides; however, many pesticides have been linked to environmental and human health concerns. Essential oils (EOs) are suitable alternatives due to their biodegradability and low toxicity to mammals. These complex mixtures of plant volatiles often display multiple biological activities and synergistic interactions between their compounds. The present work profiled the toxicity of eight EOs against the PWN in comparison to their 1:1 mixtures, to screen for successful synergistic interactions. Additionally, the main compounds of the most synergistic mixtures were characterized for their predicted environmental fate and toxicity to mammals in comparison to emamectin benzoate, a commercial nematicide used against PWN. The mixtures of Cymbopogon citratus with Mentha piperita and of Foeniculum vulgare with Satureja montana EOs showed the highest activities, with half-maximal effective concentrations (EC50) of 0.09 and 0.05 µL/mL, respectively. For these, complete PWN mortality was reached after only ca. 15 min or 2 h of direct contact, respectively. Their major compounds had a higher predicted affinity to air and water environmental compartments and are reported to have very low toxicity to mammals, with low acute oral and dermal toxicities. In comparison, emamectin benzoate showed lower nematicidal activity, a higher affinity to the soil and sediments environmental compartments and higher reported oral and dermal toxicity to mammals. Overall, uncovering synergistic activities in combinations of EOs from plants of different families may prove to be a source of biopesticides with optimized toxicity against PWNs.

Bioassays against pinewood nematode: assessment of a suitable dilution agent and screening for bioactive essential oils.

Acetone was investigated and found to be an appropriate alternative to Triton X-100 as a solvent of essential oils in bioassays aimed to investigate their effects on pinewood nematode (Bursaphelenchus xylophilus) mortality. Therefore it was used as dilution agent to screen the effectiveness of fifty two essential oils against this pest. Thirteen essential oils were highly effective, resulting in more than 90% pinewood nematode mortality at 2 mg/mL, with six of them resulting in 100% mortality. LC100 values ranged between 0.50 mg/mL and 0.83 mg/mL for the essential oils of Origanum vulgare and Satureja montana, respectively. Essential oils were submitted to gas chromatography and gas chromatography-mass spectrometry analysis and their chemical composition established. Data from essential oils with 100% mortality at 2 mg/mL and other essential oils previously found to have LC100 ≤ 2 mg/mL was combined, their chemical profiles investigated by correspondences analysis plus automatic classification.

Fungal Communities of the Pine Wilt Disease Complex: Studying the Interaction of Ophiostomatales With Bursaphelenchus xylophilus.

Considered one of the most devastating plant-parasitic nematodes worldwide, Bursaphelenchus xylophilus (commonly known as pinewood nematode, PWN) is the causal agent of the pine wilt disease in the Eurasian coniferous forests. This migratory parasitic nematode is carried by an insect vector (Monochamus spp.) into the host tree (Pinus species), where it can feed on parenchymal cells and reproduce massively, resulting in the tree wilting. In declining trees, PWN populations are strongly dependent on fungal communities colonizing the host (predominantly ophiostomatoid fungi known to cause sapwood blue-staining, the blue-stain fungi), which not only influence their development and life cycle but also the number of individuals carried by the insect vector into a new host. Our main aim is to understand if PWN-associated mycobiota plays a key role in the development of PWD, in interaction with the PWN and the insect vector, and to what extent it can be targeted to disrupt the disease cycle. For this purpose, we characterized the fungal communities of Pinus pinaster trees infected and non-infected with PWN in three collection sites in Continental Portugal with different PWD temporal incidences. Our results showed that non-infected P. pinaster mycoflora is more diverse (in terms of abundance and fungal richness) than PWN-infected pine trees in the most recent PWD foci, as opposed to the fungal communities of long-term PWD history sites. Then, due to their ecological importance for PWN survival, representatives of the main ophiostomatoid fungi isolated (Ophiostoma, Leptographium, and Graphilbum) were characterized for their adaptative response to temperature, competition in-between taxa, and as food source for PWN. Under the conditions studied, Leptographium isolates showed promising results for PWN control. They could outcompete the other species, especially O. ips, and significantly reduce the development of PWN populations when compared to Botrytis cinerea (routinely used for PWN lab culturing), suggesting this to be a natural antagonist not only for the other blue-stain species but also for the PWN.

The Fight against Plant-Parasitic Nematodes: Current Status of Bacterial and Fungal Biocontrol Agents.

Plant-parasitic nematodes (PPNs) are among the most notorious and underrated threats to food security and plant health worldwide, compromising crop yields and causing billions of dollars of losses annually. Chemical control strategies rely heavily on synthetic chemical nematicides to reduce PPN population densities, but their use is being progressively restricted due to environmental and human health concerns, so alternative control methods are urgently needed. Here, we review the potential of bacterial and fungal agents to suppress the most important PPNs, namely Aphelenchoides besseyi, Bursaphelenchus xylophilus, Ditylenchus dipsaci, Globodera spp., Heterodera spp., Meloidogyne spp., Nacobbus aberrans, Pratylenchus spp., Radopholus similis, Rotylenchulus reniformis, and Xiphinema index.

Insights into the Role of Fungi in Pine Wilt Disease.

Pine wilt disease (PWD) is a complex disease that severely affects the biodiversity and economy of Eurasian coniferous forests. Three factors are described as the main elements of the disease: the pinewood nematode (PWN) Bursaphelenchus xylophilus, the insect-vector Monochamus spp., and the host tree, mainly Pinus spp. Nonetheless, other microbial interactors have also been considered. The study of mycoflora in PWD dates back the late seventies. Culturomic studies have revealed diverse fungal communities associated with all PWD key players, composed frequently of saprophytic fungi (i.e., Aspergillus, Fusarium, Trichoderma) but also of necrotrophic pathogens associated with bark beetles, such as ophiostomatoid or blue-stain fungi. In particular, the ophiostomatoid fungi often recovered from wilted pine trees or insect pupal chambers/tunnels, are considered crucial for nematode multiplication and distribution in the host tree. Naturally occurring mycoflora, reported as possible biocontrol agents of the nematode, are also discussed in this review. This review discloses the contrasting effects of fungal communities in PWD and highlights promising fungal species as sources of PWD biocontrol in the framework of sustainable pest management actions.

Phytochemicals as Biopesticides against the Pinewood Nematode Bursaphelenchus xylophilus: A Review on Essential Oils and Their Volatiles.

The impacts of a rapidly changing environment together with the growth in global trade activities has promoted new plant pest pandemic events in forest ecosystems. The pinewood nematode (PWN), Bursaphelenchus xylophilus, causes strong worldwide economic and ecological impacts. Direct control is performed through trunk injection of powerful nematicides, however many of these (hemi)synthetic compounds have raised ecological and human health concerns for affecting non-target species and accumulating in food products. As sustainable alternatives, essential oils (EOs) have shown very promising results. In this work, available literature on the direct activity of EOs against PWN is reviewed, as a contribution to advance the search for safer and greener biopesticides to be used in sustainable PWD pest management strategies. For the first time, important parameters concerning the bioassays performed, the PWNs bioassayed, and the EOs used are summarized and comparatively analyzed. Ultimately, an overview of the chemical composition of the most active EOs allowed to uncover preliminary guidelines for anti-PWN EO efficiency. The analysis of important information on the volatile phytochemicals composing nematicidal EOs provides a solid basis to engineer sustainable biopesticides capable of controlling the PWN under an integrated pest management framework and contributes to improved forest health.

Biotransformation of menthol and geraniol by hairy root cultures of Anethum graveolens: effect on growth and volatile components.

Two oxygen-containing monoterpene substrates, menthol or geraniol (25 mg l(-1)), were added to Anethum graveolens hairy root cultures to evaluate the influence of the biotransformation capacity on growth and production of volatile compounds. Growth was assessed by the dissimilation method and by fresh and dry weight measurement. The volatiles were analyzed by GC and GC-MS. The total constitutive volatile component was composed, in more than 50%, by falcarinol (17-52%), apiole (11-24%), palmitic acid (7-16%), linoleic acid (4-9%), myristicin (4-8%) and n-octanal (2-5%). Substrate addition had no negative influence on growth. The relative amount of menthol quickly decreased 48 h after addition, and the biotransformation product menthyl acetate was concomitantly formed. Likewise, the added geraniol quickly decreased over 48 h alongside with the production of the biotransformation products. The added geraniol was biotransformed in 10 new products, the alcohols linalool, alpha-terpineol and citronellol, the aldehydes neral and geranial, the esters citronellyl, neryl and geranyl acetates and linalool and nerol oxides.

Bioactivity of Ruta graveolens and Satureja montana Essential Oils on Solanum tuberosum Hairy Roots and Solanum tuberosum Hairy Roots with Meloidogyne chitwoodi Co-cultures.

As a nematotoxics screening biotechnological system, Solanum tuberosum hairy roots (StHR) and S. tuberosum hairy roots with Meloidogyne chitwoodi co-cultures (StHR/CRKN) were evaluated, with and without the addition of the essential oils (EOs) of Satureja montana and Ruta graveolens. EOs nematotoxic and phytotoxic effects were followed weekly by evaluating nematode population density in the co-cultures as well as growth and volatile profiles of both in vitro cultures types. Growth, measured by the dissimilation method and by fresh and dry weight determination, was inhibited after EO addition. Nematode population increased in control cultures, while in EO-added cultures numbers were kept stable. In addition to each of the EOs main components, and in vitro cultures constitutive volatiles, new volatiles were detected by gas chromatography and gas chromatography coupled to mass spectrometry in both culture types. StHR with CRKN co-cultures showed to be suitable for preliminary assessment of nematotoxic EOs.

Bioactivity against Bursaphelenchus xylophilus: Nematotoxics from essential oils, essential oils fractions and decoction waters.

The Portuguese pine forest has become dangerously threatened by pine wilt disease (PWD), caused by the pinewood nematode (PWN), Bursaphelenchus xylophilus. Synthetic chemicals are the most common pesticides used against phytoparasitic nematodes but its use has negative ecological impacts. Phytochemicals may prove to be environmentally friendly alternatives. Essential oils (EOs) and decoction waters, isolated from 84 plant samples, were tested against B. xylophilus, in direct contact assays. Some successful EOs were fractionated and the fractions containing hydrocarbons or oxygen-containing molecules tested separately. Twenty EOs showed corrected mortalities ⩾96% at 2 µL/mL. These were further tested at lower concentrations. Ruta graveolens, Satureja montana and Thymbra capitata EOs showed lethal concentrations (LC100)<0.4µL/mL. Oxygen-containing molecules fractions showing corrected mortality ⩾96% did not always show LC100 values similar to the corresponding EOs, suggesting additive and/or synergistic relationships among fractions. Nine decoction waters (remaining hydrodistillation waters) revealed 100% mortality at a minimum concentration of 12.5µL/mL. R. graveolens, S. montana and T. capitata EOs are potential environmentally friendly alternatives for B. xylophilus control given their high nematotoxic properties. Nematotoxic activity of an EO should be taken in its entirety, as its different components may contribute, in distinct ways, to the overall EO activity.

Menthol and geraniol biotransformation and glycosylation capacity of Levisticum officinale hairy roots.

The biotransformation capacity of Levisticum officinale W.D.J. Koch hairy root cultures was studied by evaluating the effect of the addition of 25 mg/L menthol or geraniol on morphology, growth, and volatiles production. L. officinale hairy root cultures were maintained for 7 weeks in SH medium, in darkness at 24 degrees C and 80 r.p.m., and the substrates were added 15 days after inoculation. Growth was evaluated by measuring fresh and dry weight and by using the dissimilation method. Volatiles composition was analyzed by GC and GC-MS. Hairy roots morphology and growth were not influenced by substrate addition. No new volatiles were detected after menthol addition and, as was also the case with the control cultures, volatiles of these hairy roots were dominated by (Z)-falcarinol (1-45%), N-octanal (3-8%), palmitic acid (3-10%), and (Z)-ligustilide (2-9%). The addition of geraniol induced the production of six new volatiles: nerol/citronellol/neral (traces-15%), alpha-terpineol (0.2-3%), linalool (0.1-1.2%), and geranyl acetate (traces-2%). The relative amounts of the substrates and some of their biotransformation products decreased during the course of the experiment. Following the addition of beta-glycosidase to the remaining distillation water, analysis of the extracted volatiles showed that lovage hairy roots were able to convert both substrates and their biotransformation products into glycosidic forms. GC:gas chromatography GC-MS:gas chromatography-mass spectrometry SH:Schenk and Hildebrandt (1972) culture medium.