Endophytic mycorrhizal fungi strengthen Lactuca sativa defense against Alternaria alternata as a sustainable biocontrol approach

We established a 13‐week greenhouse experiment based on replicated microcosms to test whether the effects of defoliation on grassland plants and soil organisms depend on plant species composition and the presence of arbuscular mycorrhizal (AM) fungi. The experiment constituted of three treatment factors – plant species composition, inoculation of an AM fungus and defoliation – in a fully factorial design. Plant species composition had three levels: (1) Trifolium repens monoculture (T), (2) Phleum pratense monoculture (P) and (3) mixture of T. repens and P. pratense (T+P), while the AM inoculation and the defoliation treatment had two levels: (1) no inoculation of AM fungi and (2) inoculation of the AM fungus Glomus claroideum BEG31, and (1) no trimming, and (2) trimming of all plant material to 6 cm above the soil surface three times during the experiment, respectively. At the final harvest, AM colonization rate of plant roots differed between the plant species compositions, being on average 45% in T, 33% in T+P and 4% in P. Defoliation did not affect the colonization rate in T but raised the rate from 1% to 7% in P and from 20% to 45% in T+P. Shoot production and standing shoot and root biomass were 48%, 85% and 68% lower, respectively, in defoliated than in non‐defoliated systems, while the AM fungus did not affect shoot production and root mass but reduced harvested shoot mass by 8% in non‐defoliated systems. Of the plant quality attributes, defoliation enhanced the N concentration of harvested shoot biomass by 129% and 96% in P and T+P, respectively, but had no effect in T, while the C concentration of shoot biomass was on average 2.7% lower in defoliated than in non‐defoliated systems. Moreover, defoliation reduced shoot C yield (the combined C content of defoliated and harvested shoot biomass) on average by 47% across all plant species compositions and shoot N yield by 37% in T only. In contrast to defoliation, the AM fungus did not affect shoot N and C concentrations or shoot N yield, but induced 10% lower C yield in non‐defoliated systems and 17% higher C yield in defoliated T. In roots, defoliation led to 56% and 21% higher N concentration in P and T+P, respectively, and 28% higher C concentration in P, while the mycorrhizal fungus lowered root N concentration by 9.7% in defoliated systems and had no effect on root C concentrations. In the soil, the nematode community was dominated by bacterivores and the other trophic groups were found in a few microcosms only. Bacterivores were 45% more abundant in defoliated than in non‐defoliated systems, but were not affected by plant species composition or the AM fungus. Soil inorganic N concentration was significantly increased by defoliation in T+P, while the mycorrhizal fungus reduced NH 4 –N concentration by 40% in T. The results show that defoliation had widespread effects in our experimental systems, and while the effects on plant growth were invariably negative and those on bacterivorous nematodes invariably positive, most effects on plant C and N content and soil inorganic N concentration varied depending on the plant species present. In contrast, the effects of defoliation did not depend on the presence of the AM fungus, which suggests that while the relative abundance of legumes and grasses is likely to have a significant role in the response of legume–grass communities to defoliation, the role of AM fungi may be less important. In line with this, the AM fungus had only a few significant effects on plant and soil attributes in our systems and each of them was modified by defoliation and/or plant species composition. This suggests that the effects of AM fungi in legume–grass communities may largely depend on the plant species present and whether the plants are grazed or not.

Mycorrhizal fungi form the most important mutualistic symbioses on earth with plants. The most prevalent type of mycorrhizal fungi are the arbuscular mycorrhizal (AM) fungi. Much research has shown that the development of AM fungi is correlated with plant secondary metabolism. AM fungi can directly or indirectly affect plant secondary metabolic processes. Secondary metabolites are classified into 3 groups, terpenoids, phenolics and alkaloids. In this paper, we summarize the effects of AM fungi on the 3 groups of secondary metabolites. The relationship between terpenoids and AM fungi have been well studied, and some research has explored interactive mechanisms at the molecular level. Blumenin was first isolated and identified from mycorrhizal cereals, and its biosynthesis has been proven via the Glyceraldehyde 3_phosphate/ pyruvate pathway (MEP) by an isotopic labeling method. Since then, the accumulation of blumenin induced by AM fungi and differences in blumenin levels among different kinds of AM fungi have been observed. Studies on 1_deoxy_D_xylulose_5_phosphate synthase (DXS) and 1_deoxy_D_xylulose_5_phosphate reductoisomerase (DXR), two key enzymes in the biosynthesis of carotenoid metabolism via the MEP pathway, have found to increase the transcription of DXS and DXR in plants with AM fungi. Moreover it was temporarily and spatially correlated with the accumulation of apocarotenoids. Subsequently, two genes were identified: TC78589 encoding DXS2 which is highly expressed in roots inoculated with AM fungi, and TC77051 encoding mevalonate disphosphate decarboxylase, which is catalysed in the synthesis of terpenoids in the mevalonate pathway. Although both genes separately encode enzymes in different pathways, an enhancement of carotenoid biosynthesis has been observed. The interaction between phenolic compounds (such as phytoalexin, wall_bound phenol, flavonoids, isoflavonoids and their derivatives) and AM fungi also has been investigated intensively. It has been shown that some flavonoids stimulated the spore germination and hyphal growth of AM fungi, and the contents of flavonoids increased before the infection of AM fungi. Therefore some investigators hypothesized that flavonoids were a signal compound during the formation of AM fungi. Afterward, increased levels of flavonoids were found after the formation of AM fungi which was related to specific species of AM fungi. In addition, some experiments have indicated that the activity of peroxidase (POD), phenylalanine ammonia_lyase (PAL) and polyphenol oxidase (PPO) were significantly enhanced in AM plants. In phenylpropamoid metabolism, there are two different signaling pathways in the accumulation of secondary metabolites induced by the mycorrhizal fungus: one is through the induction of PAL and chalcone synthase (CHS), and the other is through the suppression of isoflavone reductase (IFR).Although little research seldom has examined the relationship between alkaloids and AM fungi, a recent study has shown that the formation of AM is beneficial to the accumulation of alkaloids. This study also showed the species specificity in AM affected biosynthesis of alkaloids.

Does the enhanced tolerance of arbuscular mycorrhizal plants to water deficit involve modulation of drought‐induced plant genes?

Fresh knowledge for an old relationship: new discoveries in molecular mycorrhizal research.

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Plant-driven genome selection of arbuscular mycorrhizal fungi.

Many of the disturbance‐sensitive, late successional plant species in grasslands respond to arbuscular mycorrhizal (AM) fungi more positively via growth and establishment than plants that readily establish in disturbed areas (i.e. early successional species). Inoculation with AM fungi can therefore aid the establishment of late successional species in disturbed areas. If the differential benefit of AM fungi to late versus early successional plants is context‐dependent, however, this advantage could be diminished in high phosphorus (P) post‐agricultural soils or in future climates with altered precipitation. In this greenhouse experiment, we tested if late successional plant species are less plastic in their reliance on AM fungi than early successional plants by growing 17 plant species of different successional status (9 early and 8 late successional) in full factorial combinations of inoculated or uninoculated with AM fungi, with ambient or high P levels, and with low or high levels of water. AM fungi positively affected the biomass of the 17 grassland plant species, but across all environments, late successional plant species generally responded more positively to AM fungi than early successional plants species. AM fungal growth promotion and change in below‐ground biomass allocation was generally diminished with P fertilizer across all plant species, and while there was significant variation among plant species in the sensitivity of AM fungal responsiveness to P fertilization, this differential sensitivity was not predicted by plant successional status. The role of AM fungi in plant growth promotion was not generally altered by variation in watering, however late successional plant species allocated a greater proportion of their biomass below‐ground in response to AM fungi in low versus high water conditions. Synthesis. Overall greater responsiveness to arbuscular mycorrhizal (AM) fungi by late successional species is consistent with an important role of AM fungi in plant succession, even while AM fungi are less impactful overall in high P soils. However, the increase in responsiveness of below‐ground allocation of late successional species to AM fungi in low water conditions suggests that successional dynamics may be more dependent on AM fungi in future climates that feature greater propensity for drought.

Mycorrhizal plants often have greater tolerance to drought than non-mycorrhizal plants. This study was conducted to determine the effects of arbuscular mycorrhizal (AM) fungi inoculation on total protein, essential oil content and physiological traits of Glycyrrhiza glabra grown in a greenhouse under water stress condition. The experiment was conducted in factorial arrangement in a completely randomized design using 5 replications. 100, 80, 60, 40 and 20 % of field water capacity (as water stress levels) and two AM fungal levels were applied. Control (without inoculation) and inoculated (with AM fungi) were two mycorrhizal levels. The highest and lowest leaf area values were found in AM fungi Inoculated treatment with the lowest water stress (% FC) level and 20 % FC without AM inoculation. The maximum percentage values of shoots height and shoots dry weight have been seen in in AM fungi Inoculated treatment with the lowest water stress (% FC). While the maximum percentage values of root length and root dry weight were found in AM fungi Inoculated treatment with 80 % FC water stress. Essential oil content of plants significantly increased in AM fungi Inoculated treatment with 80 % FC water stress. There were significant differences found between essential oil contents of plants at various water stress and mycorrhizal treatments. There was significant difference between total protein content of plants in AM fungi inoculated treatment with FC and 80 % FC water stress and other treatments. Total protein content was reduced from 48 to 5 µM/g FW, for AM fungi inoculated treatment with FC and without AM inoculation treatment with the 20 % FC, respectively. The results suggest that Glycyrrhiza glabra could be used for arid and semi-arid lands economical use.

James Hutton Institute, Dundee DD2 5DA, UKBelowground organisms, such as arbuscular mycorrhizal(AM) fungi, have long been credited with altering plant fit-ness. More recently, research on belowground organismshas revealed that AM fungi also influence a wide variety ofaboveground organisms via plants (reviewed in Van Dam H Bennett 2010). Schausberger et al. (2012) demon-strate that the presence of an AM fungus in the roots of ahost plant alters volatile emissions and host plant attractive-ness to parasitoids in the presence of herbivores. Thisextends previous studies that have focused on direct inter-actions of AM with plants (e.g. mycorrhizal fungal–plant–herbivore interactions; reviewed in Gehring & Bennett2009), but have not conclusively demonstrated how below-ground organisms, and AM fungi in particular, influencethird trophic level organisms such as parasitoids (Gange,Brown & Aplin 2003; Guerrieri et al. 2004; Hempel et al.2009; Leitner et al. 2010; Hoffmann, Vierheilig & Schaus-berger 2011a,b; Wooley & Paine 2011) via the release ofplant volatiles that attract parasitoids that attack herbivoreson host plants. Until recently, these studies failed to conclu-sively document the effects of AM fungi on both volatilerelease and attraction of parasitoids. For example, Wooley& Paine (2011) and Gange, Brown & Aplin (2003) haveshown variation in parasitoid attraction to plants hostingdifferent strains and species of Glomus as compared to non-mycorrhizal plants. Hoffmann, Vierheilig & Schausberger(2011a) also showed greater preference by parasitoids foreggs oviposited on plants associated with a single AMfungus. In addition, a single AM fungus in the roots of ahost plant has been shown to positively influence parasitoidlife-history characteristics (Hempel et al. 2009; Hoffmann,Vierheilig & Schausberger 2011b). However, none of thesestudies measured volatile profiles for host plants, so parasit-oid attraction could not be directly attributed to volatiles.A study on AM fungal influenced volatile release revealeddifferences but did not test whether changes in volatilesinfluenced parasitoids (Leitner et al. 2010). One study com-bined both parasitoid attractiveness and measurement ofvolatiles, but they primarily tested effects of attraction toplants in the absence of herbivory and never made compari-sons between mycorrhizal and non-mycorrhizal plantsexperiencing herbivory (Guerrieri et al. 2004). Unlike theseprevious experiments, Schausberger et al. measured bothchanges in volatile chemistry as well as parasitoid attractionin a fully factorial design.The results presented by Shausberger et al. open up multi-plefutureopportunitiesinabove–belowgroundresearch.Thefirst of these opportunities involves identifying the mecha-nisms by which AM fungi alter parasitoid attraction. Forexample,whatarethe biochemicalortranscriptionalchangesthat occur following AM fungal colonization that result inaltered volatile profiles? Are the mechanisms suggested forAM fungal alteration of direct chemical defences the samemechanisms that alter volatile profiles? Colonization by AMfungi has been shown to turn on the salicylic acid pathwaytemporarily,aprocessthatmayprimethejasmonicacidpath-way for herbivore attack (reviewed in Pozo & Azcon-Aguilar2007).Theinductionofvolatilesislinkedtothejasmonicacidpathway (reviewed in Heil 2008), and therefore, plants maybeprimedforafasterorgreaterreleaseofvolatileswhencolo-nizedbyAMfungi.However, there may be other mechanisms by which AMfungi influence volatile release. For example, given that AMfungi increase plant biomass and fitness in the PhaseolusvulgarissystemstudiedbyShausbergeret al.(aswellasmanyothersystems),itcouldsimplybethattheincreasedresourcesprovided by the mutualism allow plants to allocate moreresourcestoplantdefensivecharacteristics(e.g.directconstit-utiveandinduceddefencesaswellasindirectdefencesviavol-atile attraction; Bennett, Alers-Garcia & Bever 2006) or thatchanges in plant size or structure in association with AMfungi benefit or hinder parasitoid searching capabilities(Gange,Brown&Aplin2003).What characteristics of the volatile blends produced in thepresence of AM fungi are attractive for parasitoids? Shaus-bergeret al.showedtherewerefewerchemicalspresentinthevolatileblendsofAMfungalplantsbeforeherbivory(relativeto plants not hosting AM fungi), but this difference disap-peared after herbivory. However, different volatile chemicalswere released from plants experiencing herbivory and colo-nizedornotbyAMfungi(seealsoLeitneret al.2010).Shaus-berger et al. did not address whether increased attraction toplants hosting AM fungi is associated with a particular vola-

A glimpse into the past of land plants and of their mycorrhizal affairs: from fossils to evo‐devo

Thai Jasmine rice, a high-quality variety of rice, can further be enhanced for the economic values through organic cultivation using arbuscular mycorrhizae fungi. Therefore, a study was conducted during 2017 to 2019 at Chiang Mai University and Tin Nee Yom Farm in Chiang Mai province, Thailand. This study investigated the optimum substrate formulas and the host plants for inoculum production of arbuscular mycorrhizal (AM) fungi, and to evaluate the efficiency of the inoculum of AM fungi for the growth and yield of Thai Jasmine rice in paddy fields of organic farming. The substrates used for in oculum production were soil, sand, leaf compost and vermiculite. AM fungi were cultured in pots of eight substrate formulas and used Brachiaria ruziziensis and Crotalaria juncea as the host plants. The results showed that the best formula which had the highest AM spore density was the formula containing compost mixed with vermiculite (1:2 v/v) and used B. ruziziensis as the host plant. For using C. juncea as the host plant, the optimum formula for spore production of AM fungi the formula containing soil mixed with vermiculite at the ratio of 2:1 (v/v). The dominant species of AM fungi associated with B. ruziziensis in the optimum formula were found to be four species of Aaulospora scrobiculata, A. tuberculata, Funnelifomis coronatum and Rhizophagus intreradices. Whereas C. juncea was used as the host plant in the optimum formula, there were also found to be three species of the AM fungi except R. intreradices. Using of AM fungi from the highest inoculum product as a biofertilizer in the organic farming in Chiang Dao district, Chiang Mai province in northern Thailand, increased growth of Thai Jasmine rice by about 12% in the paddy field without compost application and about 13% with compost application to the rice plants. N, P and K contents in the rice grains were about 1.3, 1.5 and 6.9 times higher than those of the uninoculated treatment. Therefore, the AM inoculum product of this study can be used as an efficient biofertilizer for increasing grain yields of Thai Jasmine rice in organic paddy fields. Both of optimum formulas can be used for commercial inoculum production of AM fungi for using as biofertilizer in organic farming.

In natural and agricultural ecosystems, arbuscular mycorrhizal (AM) fungi play a major role in plant nutrition. In AM symbiosis, the AM fungi extract mineral nutrients from the substrate and transfer them to the host plant. Inside the roots of the host plant, the intraradical hyphae form tree like structures (arbuscules) where the nutrients are released to the plant fungal interface. In return, the AM fungi receive carbohydrates from the plants. Specialized transport systems enable nutrient uptake from the substrate and translocation across membranes. As main components of organic molecules, phosphorus (P), nitrogen (N) and carbon (C) are of particular importance for symbiotic nutrient exchanges. This work is focused on a range of genes that encode proteins contributing to transport molecules (P, N and C nutrients) across cellular membranes in the plants Populus trichocarpa (poplar) and Sorghum bicolor (sorghum), and in the AM fungus Rhizophagus irregularis. In the AM fungus R. irregularis (formerly Glomus intraradices), we identified and characterized a novel functional ammonium transporter (AMT), GintAMT3. Quantification of transcript abundances in the extraradical mycelium (ERM) and the intraradical mycelium (IRM) during symbiosis with poplar and sorghum revealed that GintAMT3 was highly expressed in the IRM of AM roots. Phylogenetic analysis showed further, that the six glomeromycotan AMTs share high sequence similarity, but are distinct to AMTs of other fungal phyla. To functionaly analyze GintAMT3, we expressed GintAMT3 in a yeast deletion mutant devoid of all AMTs. The heterologous expression revealed that GintAMT3 is a low affinity transporter. Heterologous expression of GFP tagged GintAMT3 in yeast showed that GintAMT3 is localized in the plasma membrane and the vacuolar membrane. Further, we could show that expression of GinAMT3 is dependent on the N nutrition status and the fungal C status. Taken together, our data suggested that GintAMT3 is the main export carrier for ammonium at the arbuscular site. Using mRNA sequencing, we could show that low N availability significantly increased gene expression of the AM fungus, including genes involved in cell growth and membrane biogenesis as well as genes involved in signaling and metabolic processes. High abundances of genes related to N metabolism, including glutamine synthase, aminotransferase, AMTs as well as arginases, indicated a high turnover rate of N in the symbiotic root tissue. Depending on P availability, gene expression of AM phosphate transporters (PT) and AMT changed. Induction of PT and AMT under low-P availability indicated that the AM fungus transfers more nutrients to the host plant. Further, we identified amino acids transporters and H+/oligopeptide transporters specifically induced in mycorrhizal poplar roots, indicating that amino acids are transferred between the AM fungus and the plant. In poplar, we found that root colonization and low-N conditions resulted in the down-regulation of defense gene expression, suggesting that the plant stimulated symbiotic interactions with the AM fungus. We showed that root colonization specifically induced expression of known and newly identified PT and AMT in poplar and sorghum. Specific induction of nutrient transporters upon starvation strongly indicated that they are essential components of a functional symbiosis and suggested they are located in AM roots. Furthermore, root colonization suppressed the expression of genes involved in P starvation response, indicating that root colonization efficiently alleviated P stress of the plant. Moreover, we could show that the annual sorghum is more dependent on the AM fungus than the perennial poplar, but also that more P and possibly also more N is transferred from the AM fungus to the host plant. Non-mycorrhized sorghum accumulated similar quantities of P as AM sorghum under conditions, in which only the AM fungus had access to the P source. Poplar on the other hand accumulated less P in AM plants. In addition, we observed that a subset of poplar Pht1 transporters was regulated independently on the AM fungus, but depending on the P availability of the substrate. To deepen our understanding about symbiotic C exchange, we made transcriptome analysis and qRT-PCR to investigate the role of carbohydrate transporters in AM symbiosis between R. irregularis and, poplar and sorghum, respectively. In R. irregularis, the monosaccharide transporter GintMST2 was specifically induced in the IRM independently on the nutrient condition. Interestingly, we observed the down-regulation of many carbohydrate transporters in AM roots of poplar and sorghum. However, in poplar, we identified one carbohydrate transporter, which might be involved in symbiotic C transfer. In conclusion, our data on C transport suggested that carbohydrates are taken from the plant by the AM fungus instead of actively transferred to the fungus by the host plant. Taken together, the data summarized in my thesis add to our understanding of nutrient transport in AM symbiosis under different environmental conditions and help elucidating the underlying mechanisms. Regarding climate changes and resources shortening, a precise understanding of the efficiency of AM symbiosis may help to increase the efficiency of sustainable agriculture.

Arbuscular mycorrhizal (AM) fungi reside in the rhizosphere and form mutualistic associations with plant roots. They promote photosynthesis, improve stress resistance, and induce secondary metabolite biosynthesis in host medicinal plants. The AM fungi that are symbiotic with medicinal plants comprise a wide array of species and have abundant germplasm resources. Though research on the AM fungi in medicinal plants began relatively recently, it has nonetheless become an investigative hot spot. Several scholars have explored the diversity and the growth-promoting effects of mycorrhizal fungi in hundreds of medicinal plants. Current research on symbiotic AM fungi in medicinal plants has focused mainly on the effects of inoculating host plants with symbiotic mycorrhizal fungi. However, research on the symbiotic AM fungi in medicinal plants continues to expand, and further study is required to determine the mechanisms by which AM fungi interact with host plants. This paper introduces the diversity of symbiotic AM fungi of medicinal plants and the effects of AM fungi on rhizosphere soil of medicinal plants, including soil structure, microbiota, enzyme activities, etc. This review focuses on the effects of AM fungi on medicinal plants, including antioxidant enzyme systems, drought resistance, nutrient absorption profiles of macro- and micronutrients, accumulation of secondary metabolites such as terpenes, phenolic compounds, and nitrogenous compounds, and prevention of diseases. This review is expected to provide a reference for the application of AM fungi in medicinal plant cultivation, biological control, resource conservation, and the sustainable development of the traditional Chinese medicine industry.

Interactions between phosphorus availability and an AM fungus ( Glomus intraradices) and their effects on soil microbial respiration, biomass and enzyme activities in a calcareous soil

Over 80% of vascular plant species are associated with some type of mycorrhizal fungus (Trappe 1987). These associations are thought to be mutualistic in many cases, with the host plant exchanging photosynthate with the fungus in return for mineral nutrients (Harley and Smith 1983, Smith and Read 1997). The two most abundant and studied mycorrhizal types are ectomycorrhizal (EM) fungi and arbuscular mycorrhizal (AM) fungi. A generally agreed-upon phenomenon is that AM fungi are broad generalists with respect to the number of host plant species with which they can associate (Smith and Read 1997). Because of the difficulty of detecting variation in the relative abundances of AM fungal species on plant roots, as well as the difficulty of understanding the causes (environmental factors, host genotype, interspecific interactions) of such variation, there exist gaps in our knowledge about the extent to which AM fungal species associate with different plant species, especially in complex natural fungal communities. However, based on evidence accumulated so far from field and laboratory observations, AM fungi exhibit broad host ranges, and the rare exceptions to this (e.g., Graw et al. 1979) must not be allowed to cloud the observation that VA mycorrhizal fungi and their host plants have generally non-specific (Smith and Read 1997). In contrast, specificity among EM fungi ranges from very broad to extremely specific, with many EM fungi associating with only a single host plant species (Janos 1 980a, Alexander 1989, Harley 1989, Borowicz and Juliano 1991, Molina et al. 1992, Smith and Read 1997). Explanations for the disparity in host specificity between the two types of fungi have been mentioned briefly in other contexts, but have not been developed, tested, or compared with other potential explanations (e.g., Janos 1980b, 1983, Malloch et al. 1980, Connell and Lowman 1989, Molina et al. 1992). There is clearly a need to explicitly develop and test a set of hypotheses explaining the higher incidence of host-specificity among EM fungi than among AM fungi. The general topic of specificity is one of broad interest to ecologists and evolutionary biologists, as the degree of specificity in interactions is predicted to strongly influence the nature of evolution of those interactions (e.g., whether two species co-evolve) (Janzen 1980, Thompson 1994). As a result, it has received a great deal of attention both theoretically and empirically. Much of this work has focused on specificity in interactions between plants and their herbivores (e.g., Futuyma and Moreno 1988, Berenbaum 1996), pollinators (e.g., Kiester et al. 1984, Waser et al. 1996), and fruit dispersers (e.g., Jordano 1987, Fleming et al. 1993). The voluminous literature generated from investigations of these three types of interactions provides valuable information and predictions about specificity phenomena. Many of these predictions are particular to specific systems, but some are very general and could aid in attempts to understand specificity in mycorrhizal mutualisms. However, these results have not been utilized to help understand the disparity in host specificity between AM and EM fungi. Here I discuss three principles regarding the evolution of specificity that emerge from an examination of the plant-herbivore, plant-pollinator, and plant-fruit-disperser literature and other theoretical literature. These three principles suggest three hypotheses (not mutually exclusive) for the disparity in host specificity between AM and EM fungi, which I discuss and evaluate.