AMF Inoculation Can Enhance Yield of Transgenic Bt Maize and Its Control Efficiency Against Mythimna separata Especially Under Elevated CO2.

BackgroundThe cultivation of Bt maize (maize genetically modified with Bacillus thuringiensis) continues to expand globally. Arbuscular mycorrhizal fungi (AMF), an important kind of microorganism closely related to soil fertility and plant nutrition, may influence the ecological risk of target lepidopteran pests in Bt crops.MethodsIn this study, transgenic Bt maize (Line IE09S034 with Cry1Ie vs. its parental line of non-Bt maize cv. Xianyu335) was inoculated with a species of AMF, Glomus caledonium (GC). Its effects on the food utilization, reproduction and development of armyworm, Mythimna separata, were studied in a potted experiment from 2017 to 2018.ResultsGC inoculation increased the AMF colonization of both modified and non-modified maize, and also increased the grain weight per plant and 1,000-grain weight of modified and non-modified maize. However, the cultivation of Bt maize did not significantly affect the AMF colonization. The feeding of M. separata with Bt maize resulted in a notable decrease in RCR (relative consumption rate), RGR (relative growth rate), AD (approximate digestibility), ECD (efficiency of conversion of digested food) and ECI (efficiency of conversion of ingested food) parameters in comparison to those observed in larvae fed with non-Bt maize in 2017 and 2018, regardless of GC inoculation. Furthermore, remarkable prolongation of larval life span and decreases in the rate of pupation, weight of pupa, rate of eclosion, fecundity and adult longevity of M. separata were observed in the Bt treatment regardless of GC inoculation during the two-year experiment. Also, when M. separata was fed with Bt maize, a significant prolongation of larval life and significant decreases in the pupal weight, fecundity and adult longevity of M. separata were observed when inoculated with GC. However, it was just the opposite for larvae fed with non-Bt maize that was inoculated with GC. The increased percentage of larval life-span, the decreased percentages of the food utilization, and the other indexes of reproduction, growth, and development of M. separata fed on Bt maize relative to non-Bt maize were all visibly lower when under GC inoculation in contrast to the CK.DiscussionIt is presumed that Bt maize has a marked adverse impact on M. separata development, reproduction and feeding, especially when in combination with the GC inoculation. Additionally, GC inoculation favors the effectiveness of Bt maize against M. separata larvae by reducing their food utilization ability, which negatively affects the development and reproduction of the armyworm. Thus, Bt maize inoculated with AMF (here, GC) can reduce the severe threats arising of armyworms, and hence the AMF inoculation may play an important ecological functions in the field of Bt maize ecosystem, with potentially high control efficiency for the target lepidopteran pests.

My dissertation research examined the effect of the cultivation of insect-resistant Bacillus thuringiensis (Bt) maize on the soil environment with a goal of understanding how to obtain a balance between technological advancement and maintenance of a healthy soil ecosystem. Although Bt plants may help to reduce pesticide use, conferring benefits to farm workers and the environment, there are still unresolved questions about how the cultivation of Bt plants affects soil organisms. For this dissertation project, I used 14 different genotypes of Bt maize and non-Bt maize (Zea mays) to investigate the effects of transgenic Bt plants on the colonization ability, abundance, and diversity of symbiotic arbuscular mycorrhizal fungi (AMF) in the soil ecosystem over time. My greenhouse studies demonstrated that Bt maize plants exhibited reduced AMF colonization across multiple Bt genotypes and that effects were most pronounced when fertilizer levels were limited and spore density was high. In addition, I found that although differences in AMF colonization between Bt and non-Bt maize were difficult to detect in the field, spore density was reduced in Bt field plots after just one growing season. When I tested the effect of plot history on AMF and plant growth, I found that Bt and non-Bt maize plants had higher leaf chlorophyll content when grown in plots previously cultivated with the same maize line as the previous year, indicative of a positive feedback effect. I also examined potential mechanisms contributing to the reduced AMF colonization observed in Bt maize in greenhouse studies and determined that follow-up experiments should continue to investigate differences in root apoplastic invertase activity and root permeability in Bt and non-Bt maize. Future investigations would also benefit from examining potential differences in root exudate profiles and volatile organic compounds between Bt and non-Bt cultivars. Taken together, my dissertation results suggest that, while difficult to detect in the field, reductions in AMF colonization in Bt maize roots may be ecologically significant as they could lead to a decrease in the abundance of AMF propagules in the soil over time, potentially impacting soil structure and function in areas where Bt crop cultivation is high.

Background: The cultivation of Bt maize (maize genetically modified with Bacillus thuringiensis) continues to expand globally. Arbuscular mycorrhizal fungi (AMF), a kind of beneficially microbial community, closely related to soil fertility and plant nutrition, may influence the resistance risk of Bt crops against target lepidopteran pests. Methods: In this study, transgenic Bt maize (Line IE09S034 with Cry1Ie vs. its parental line of non-Bt maize cv. Xianyu335) was inoculated with a species of AMF, Glomus caledonium (GC) and its outcomes on the food utilization, reproduction and development of armyworm, Mythimna separata were conducted in a potted experiment from 2017 to 2018. Results: AMF inoculation showed favorable influence on the GC colonization of both modified and non-modified maize, and marked increase in the grain weight per plant and 1000-grain weight of modified and non-modified maize. Meanwhile, the cultivation of Bt maize didn’t significantly affected the AMF colonization. The feeding of M. separata with Bt maize resulted in a notable decrease in the parameters RCR, RGR, AD, ECI and ECD as compared with those observed in larvae fed with non-Bt maize in 2017 and 2018 regardless of GC inoculation. Furthermore, remarkable shortening of the adult longevity, remarkable prolongation of larval life span and remarkable decrease in the rate of pupation, weight of pupa, rate of eclosion and fecundity of M. separata was observed in Bt treatment regardless of GC inoculation during the two year experiment. Also, when M. separata was given Bt, a significant prolongation of larval life and shortening of the adult longevity, and a significant decrease of the pupal weight and fecundity of M. separate, was observed when inoculated with GC. However, it was just the opposite for larvae fed with non-Bt maize and inoculated with GC. The increased percentage of larval life-span, and the decrease percentages of the food utilization and the other indexes of reproduction, growth, and development of M. separata fed on Bt maize relative to non-Bt maize were all obviously lower under GC inoculation in contrast to the CK. Discussion: It is presumed that Bt maize has marked adverse impact on the M. separata development, reproduction and feeding, especially in combination with the GC inoculation. And the GC inoculation favors the resistance efficiency of Bt maize against M. separata larvae through reducing their food utilization ability, and then negatively affecting the development and reproduction of the armyworm. So Bt maize inoculated with AMF (here, GC) can lessen the severe threats arising from armyworms, and hence the AMF inoculation may play important ecological functions in the field of Bt maize ecosystem with potentially high control efficiency for the target lepidopteran pests.

The cultivation of genetically engineered Bacillus thuringiensis toxin-expressing (Bt) maize continues to increase worldwide, yet the effects of Bt crops on arbuscular mycorrhizal fungi (AMF) in soil are poorly understood. In this field experiment, we investigated the impact of seven different genotypes of Bt maize and five corresponding non-Bt parental cultivars on AMF and evaluated plant growth responses at three different physiological time points. Plants were harvested 60 days (active growth), 90 days (tasseling and starting to produce ears), and 130 days (maturity) after sowing, and data on plant growth responses and percent AMF colonization of roots at each harvest were collected. Spore abundance and diversity were also evaluated at the beginning and end of the field season to determine whether the cultivation of Bt maize had a negative effect on AMF propagules in the soil. Plant growth and AMF colonization did not differ between Bt and non-Bt maize at any harvest period, but AMF colonization was positively correlated with leaf chlorophyll content at the 130-day harvest. Cultivation of Bt maize had no effect on spore abundance and diversity in Bt versus non-Bt plots over one field season. Plot had the most significant effect on total spore counts, indicating spatial heterogeneity in the field. Although previous greenhouse studies demonstrated that AMF colonization was lower in some Bt maize lines, our field study did not yield the same results, suggesting that the cultivation of Bt maize may not have an impact on AMF in the soil ecosystem under field conditions.

Simple SummaryBt crops are a key strategy for controlling Spodoptera exigua (Hübner), yet the behavioral characteristics of this pest on Bt and non-Bt plants remain unexplored. This study demonstrates that S. exigua larvae prefer to feed on non-Bt maize and exhibit antifeedant and avoidance behavior toward Bt maize expressing Cry1Ab + Vip3Aa19 proteins. Although female moths showed no oviposition preference between Bt and non-Bt maize plants under undamaged conditions, they preferentially oviposited on Bt maize when non-Bt maize plants were damaged. Under the seed-mixture refuge pattern, S. exigua larvae exhibited frequent interplant movement between Bt and non-Bt maize plants. Increasing the proportion of non-Bt maize significantly enhanced larval dispersal distances and raised the risk of transit damage to Bt maize plants. These findings clarify the behavioral responses of S. exigua to Bt and non-Bt maize plants, and provide scientific evidence for optimizing refuge strategy to delay resistance evolution.Establishing refuges is a primary strategy for managing resistance in target pests against Bt maize. The larval feeding and dispersal, and adult oviposition behaviors of Spodoptera exigua (Hübner) on Bt and non-Bt maize plants are critical factors in determining optimal refuge configurations. This study employed laboratory and field experiments to evaluate the larval feeding and dispersal behaviors, as well as the oviposition preferences of S. exigua moths, on Bt (Cry1Ab + Vip3Aa19) and non-Bt maize plants. Results showed that as time of the choice test increased, the larval selection rate on Bt maize leaves declined progressively, with all instars (1st–5th) preferring to feed on non-Bt maize. After 48 h, the selection rates of larvae for non-Bt and Bt maize were 40.63–66.25% and 9.38–33.75%, respectively. Female moths exhibited no significant oviposition preference between Bt and non-Bt plants under undamaged conditions; however, when non-Bt maize was infested by the larvae, females preferentially oviposited on Bt maize plants (73.55%). Under the seed-mixture refuge pattern in field conditions, increasing the proportion of non-Bt maize significantly enhanced larval dispersal distances and facilitated larval transit damage between Bt and non-Bt plants. Our research clarifies the behavioral patterns of S. exigua on Bt and non-Bt maize, provides a scientific basis for optimizing refuge strategy to delay the development of resistance.

Arbuscular mycorrhizal (AM) fungal diversity of arid lands : from AM fungal species to AM fungal communities

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-

Effect of Bacillus thuringiensis (Bt) maize cultivation history on arbuscular mycorrhizal fungal colonization, spore abundance and diversity, and plant growth

Insect-resistant Bacillus thuringiensis (Bt) maize is widely cultivated, yet few studies have examined the interaction of symbiotic arbuscular mycorrhizal fungi (AMF) with different lines of Bt maize. As obligate symbionts, AMF may be sensitive to genetic changes within a plant host. Previous evaluations of the impact of Bt crops on AMF have been inconsistent, and because most studies were conducted under disparate experimental conditions, the results are difficult to compare. We evaluate AMF colonization in nine Bt maize lines, differing in number and type of engineered trait, and five corresponding near-isogenic parental (P) base hybrids in greenhouse microcosms. Plants were grown in 50% local agricultural soil with low levels of fertilization, and AMF colonization was evaluated at 60 and 100 d. Nontarget effects of Bt cultivation on AMF colonization were tested in a subsequently planted crop, Glycine max, which was seeded into soil that had been preconditioned for 60 d with Bt or P maize. We found that Bt maize had lower levels of AMF colonization in their roots than did the non-Bt parental lines. However, reductions in AMF colonization were not related to the expression of a particular Bt protein. There was no difference in AMF colonization in G. max grown in the Bt- or P-preconditioned soil. These findings are the first demonstration of a reduction in AMF colonization in multiple Bt maize lines grown under the same experimental conditions and contribute to the growing body of knowledge examining the unanticipated effects of Bt crop cultivation on nontarget soil organisms.

The ubiquitous symbiosis between plants and arbuscular mycorrhizal (AM) fungi is multifunctional. In this symbiosis, plants exchange photosynthates for phosphorus (P) and other mineral nutrients, and they gain increased resistance to soil borne diseases, drought and extreme temperature. All of these benefits might be crucial for plants growing in extreme environments. The aim of this thesis was to shed light on the diversity and dynamics of AM fungal communities in Southern Arabia, known for its particularly arid conditions and low fertility of soils. AM fungal communities in two agricultural sites were compared with those in adjacent natural habitats. The agricultural sites were cultivated with date palms (Phoenix dactylifera) and managed according to “traditional” and “modern” farming systems. The natural sites contained native plant species (among those Zygophyllum hamiense, Salvadora persica, Prosopis cineraria and Heliotropium kotschyi). Soil was sampled from the rhizosphere of plants and from these samples, AM fungal spores were isolated and morphologically identified. Furthermore, “trap cultures” were established in the green house, using the soil samples from the field as AM fungal inocula. The results showed that the AM fungal community composition at the agricultural sites differed from that at the natural habitats. Agricultural sites had a much higher AM fungal spore abundance, species richness and inoculum potential supposedly due to the land-use change from natural to agricultural with irrigation and fertilizer application. A molecular approach was used to identify the AM fungi colonizing the roots of the date palms at the two agricultural sites. Nine phylogenetic taxa were revealed, eight of which could be attributed to the Glomus group A, the most diverse group in the Glomeromycota, and one to the Scutellospora group that occurred at the traditional agriculture site only. Two of the nine taxa could be associated to AM fungal species already described. These were Glomus sinuosum and Glomus proliferum. Three phylotype groups were associated with AM fungal sequences previously detected in environmental samples. The other 4 phylotype groups were not associated with any of the sequences in the GenBank nor in large database of the Botanical Institute and, therefore, we assume that they are new to science. The communities of these fungi were found to differ between the two agricultural sites and consisted of both site-specialist and site-generalist groups. This was in accordance with spore morphospecies differences found between the two sites. The composition of the detected phylotypes was quite unique because it lacked certain groups commonly occurring in most habitats around the world investigated so far. Trap cultures inoculated with rhizosphere soils of date palms growing on a modern agricultural plantation showed an AM fungal community consisting of Glomus aurantium, Glomus intraradices, Diversispora spurca, Acaulospora sp. and five different Glomus phylotypes which presumably new to science. Based on morphological identification of AM fungal spores, a total of 36 morphospecies were detected at the five sites investigated in Southern Arabia. Twenty two of them belonged to the genus Glomus, six to Scutellospora, four to Acaulospora, two to Archaeospora and one to each genus of Paraglomus and Ambispora. This is a quite high richness considering that so far only around 200 AM fungal species have been described worldwide in the phylum Glomeromycota. The composition of AM fungal communities detected in this study was compared with communities found in other habitats of the world to seek for biogeographical patterns. It was found that the agricultural sites in the present study have a composition most similar to those 2 found at sites with sandy soils around the world. The natural sites, however, seem to maintain a unique species composition, which might have emerged due to unique local biotic and abiotic environmental factors of Southern Arabia. To my knowledge, this is the first report on AM fungal communities in Arabian Peninsula and the first molecular investigation ever on AM fungi associated with date palm, a socioeconomically important plant in many dry lands of the world. On a global scale, I believe that this work is a significant contribution to the knowledge on diversity, phylogeny and ecology of AM fungi.

The cultivation of transgenic Bacillus thuringiensis (Bt) has received worldwide attention since Bt crops were first released. Its ecological risks on arbuscular mycorrhizal fungi (AMF) have been widely studied. In this study, after cultivation for five seasons, the AMF diversity and community composition of two Bt maize varieties, 5422Bt1 (event Bt11) and 5422CBCL (event MO10), which both express Cry1Ab protein, and their isoline non-Bt maize 5422, as well as Bt straw after cultivation had been returned to subsequent conventional maize variety, were analyzed using Illumina MiSeq sequencing. A total of 263 OTUs (operational taxonomic units) from 511,847 sequenced affiliated with the AMF which belonged to Mucoromycota phylum Glomeromycotina subphylum were obtained. No significant difference was detected in the AMF diversity and richness (Shannon, Simpson, ACE, and Chao 1 indices) and community composition in rhizosphere soils and roots between Bt and non-Bt treatment revealed by NMDS (non-metric multidimensional scaling) and NPMANOVA (non-parametric multivariate analysis). Moreover, Glomus was the most dominant genus in all samples. Although there was no significant difference in the AMF community in roots and rhizosphere soils between the Bt and non-Bt maize treatments, total phosphorus (TP), total nitrogen (TN), organic carbon (OC), and pH were driving factors affecting the AMF community, and their composition varied between rhizosphere soils and roots during the maturity period of the fifth season. Compared to our previous study, the results were identical. In conclusion, no significant difference was observed between the Bt and non-Bt treatments, and the Illumina MiSeq method had higher throughput and higher quality read cover, which gave us comprehensive insight into AMF communities in agro-ecosystems.

The cultivation of genetically modified plants (GMP) has raised concerns regarding the plants’ ecological safety. A greenhouse experiment was conducted to assess the impact of five seasons of continuous Bt (Bacillus thuringiensis) maize cultivation on the colonisation and community structure of the non-target organisms arbuscular mycorrhizal fungi (AMF) in the maize roots, bulk soils and rhizospheric soils using the terminal restriction fragment length polymorphism (T-RFLP) analysis of the 28S ribosomal DNA and sequencing methods. AMF colonisation was significantly higher in the two Bt maize lines that express Cry1Ab, 5422Bt1 (event Bt11) and 5422CBCL (MON810) than in the non-Bt isoline 5422. No significant differences were observed in the diversity of the AMF community between the roots, bulk soils and rhizospheric soils of the Bt and non-Bt maize cultivars. The AMF genus Glomus was dominant in most of the samples, as detected by DNA sequencing. A clustering analysis based on the DNA sequence data suggested that the sample types (i.e., the samples from the roots, bulk soils or rhizospheric soils) might have greater influence on the AMF community phylotypes than the maize cultivars. This study indicated that the Cry1Ab protein has minor effects on the AMF communities after five seasons of continuous Bt maize cultivation.

Efficacy of arbuscular mycorrhizal (AM) inoculum and compost application on cultivation of cassava variety Hanatee (a sweet type) under organic farming are required to investigate for management in order to obtain high yield and quality of the cassava product. Therefore, an experiment was conducted during 2018 to 2020 at organic farm in Chiang Mai province, northern Thailand to examine the efficacy of inoculum product of AM fungi and leafcompost application on the growth, tuber yield and nutrient uptake of the cassava variety. Dominant species of AM fungi isolated from inoculum product (Aaulospora scrobiculataand A. tuberculata) were used for the study and sunn hemp acted as host plant for inoculum production. Four experimental treatments viz., control (without application of boththe AM fungal inoculum and compost), application of AM fungal inoculum without compost, application of compost without AM fungal inoculum, and applications of both AM fungal inoculum and compost were laid out in randomized block design with four replications. Nine months of cassava cultivation, the results showed that application of AM fungal inoculum increased growth, tuber yield and nutrient uptake of cassava variety Hanatee, especially the higher shoot dry weight (153.4 g/plant), tuber dry weight (229.3 g/plant), N andP contents in tuber (321.0 and 255.3 mg/plant, respectively) were recorded when inoculum of AM fungiwas used in combination with the compost application. Tuber dry weights of cassava in thetreatments applied only AM fungal inoculum, applied only compost, and applied both AM fungal inoculum in combination with compost were about 1.7, 5.1 and 11.4 times higher than the control treatment, respectively. Furthermore, compost application had positive impact on root colonization and spore densities of AM fungi in the cassava field. Therefore, this study highlights the use of AM fungi inoculum together with compost application for a move towards sustainable organic farming.

Symbiotic interactions between arbuscular mycorrhizal (AM) fungi and male papaya plants: Its status, role and implications

Diversity and efficacy of arbuscular mycorrhizal (AM) fungi isolated from soils of soybean fields