Congruence of fatty acid methyl ester profiles and morphological characters of arbuscular mycorrhizal fungi in Gigasporaceae.

Arbuscular mycorrhizal (AM) fungi (Order Glomales, Class Zygomycetes) are a diverse group of soil fungi that form mutualistic associations with the roots of most species of higher plants. Despite intensive study over the past 25 years, the phylogenetic relationships among AM fungi, and thus many details of evolution of the symbiosis, remain unclear. Cladistic analysis was performed on fatty acid methyl ester (FAME) profiles of 15 species in Gigaspora and Scutellospora (family Gigasporaceae) by using a restricted maximum likelihood approach of continuous character data. Results were compared to a parsimony analysis of spore morphological characters of the same species. Only one tree was generated from each character set. Morphological and developmental data suggest that species with the simplest spore types are ancestral whereas those with complicated inner wall structures are derived. Spores of those species having a complex wall structure pass through stages of development identical to the mature stages of simpler spores, suggesting a pattern of classical Haeckelian recapitulation in evolution of spore characters. Analysis of FAME profiles supported this hypothesis when Glomus leptotichum was used as the outgroup. However, when Glomus etunicatum was chosen as the outgroup, the polarity of the entire tree was reversed. Our results suggest that FAME profiles contain useful information and provide independent criteria for generating phylogenetic hypotheses in AM fungi. The maximum likelihood approach to analyzing FAME profiles also may prove useful for many other groups of organisms in which profiles are empirically shown to be stable and heritable.

Glomus intraradices and Gigaspora margarita arbuscular mycorrhizal associations differentially affect nitrogen and potassium nutrition of Plantago lanceolata in a low fertility dune soil

Two controlled microcosm experiments aimed at a critical re-assessment of the contributions of divergent arbuscular mycorrhizal (AM) fungi to plant mineral nutrition were established that specifically targeted Plantago lanceolata–Glomus intraradices (B.B/E) and –Gigaspora margarita (BEG 34) symbioses developed in a native, nutrient limited, coastal dune soil. Plant tissue nitrogen (N), phosphorus (P) and potassium (K) status as well as plant growth parameters and levels of mycorrhizal colonization were assessed at harvest. In addition to the general well-established mycorrhizal facilitation of P uptake, the study was able to demonstrate a G. intraradices-specific contribution to improved plant nitrogen and potassium nutrition. In the two respective experiments, G. intraradices-inoculated plants had 27.8% and 40.8% more total N and 55.8% and 23.3% more total K when compared to Gi. margarita inoculated counterparts. Dissimilar overall contribution of the two isolates to plant nutrition was identified in AM-genus specific differences in plant tissue N:P:K ratios. G. intraradices inoculated and non-mycorrhizal plants generally exhibited N:P:K ratios indicative of P limitation whereas for Gi.margarita mycorrhizal plants, corresponding ratios strongly implied either N or K limitation. The study provides further evidence highlighting AM functional biodiversity in respect to plant nutrient limitation experienced by mycorrhizal P. lanceolata in an ecologically relevant soil system.

Arbuscular mycorrhizal modulation of diazotrophic and denitrifying microbial communities in the (mycor)rhizosphere of Plantago lanceolata

Impacts of divergent arbuscular mycorrhizal (AM) fungi, Glomus intraradices and Gigaspora margarita, on denitrifying and diazotrophic bacterial communities of Plantago lanceolata in nutrient-limited dune soil were assessed. We hypothesized AM species-related modifications that were confirmed in respective bacterial nirK and nifH sequence polymorphism -based community clustering and community variance allocation. The denitrifying community appeared more responsive to AM fungi than the nitrogen-fixing community. Nevertheless, the main explanatory variable, in both cases, was plant age. We conclude that AM fungi can modify N-cycling microbial rhizosphere communities and future work should aim to verify the functional significance and mechanistic basis.

Contrasting arbuscular mycorrhizal communities colonizing different host plants show a similar response to a soil phosphorus concentration gradient

High soil phosphorus (P) concentration is frequently shown to reduce root colonization by arbuscular mycorrhizal (AM) fungi, but the influence of P on the diversity of colonizing AM fungi is uncertain. We used terminal restriction fragment length polymorphism (T-RFLP) of 18S rDNA and cloning to assess diversity of AM fungi colonizing maize (Zea mays), soybean (Glycene max) and field violet (Viola arvensis) at three time points in one season along a P gradient of 10280mgl1 in the field. Percentage AM colonization changed between sampling time points but was not reduced by high soil P except in maize. There was no significant difference in AM diversity between sampling time points. Diversity was reduced at concentrations of P > 25mgl1, particularly in maize and soybean. Both cloning and T-RFLP indicated differences between AM communities in the different host species. Host species was more important than soil P in determining the AM community, except at the highest P concentration. Our results show that the impact of soil P on the diversity of AM fungi colonizing plants was broadly similar, despite the fact that different plants contained different communities. However, subtle differences in the response of the AM community in each host were evident.

Heterogeneity in inoculum potential and effectiveness of arbuscular mycorrhizal fungi

Arbuscular mycorrhizae are symbiotic associations among glomalean fungi and plant roots that often lead to enhanced water and nutrient uptake and plant growth. We describe experiments to test whether inoculum potential of arbuscular mycorrhizal (AM) fungal communities varies spatially within a broadleaf temperate forest, and also whether there is variability in the effectiveness of AM fungal communities in enhancing seedling growth. Inoculum potential of arbuscular mycorrhizal fungi in a temperate broad-leaved forest did not vary significantly among sites. Inoculum potential, measured as the extent to which the roots of red maple seedlings that had been germinated on sterile sand and then transplanted into the forest, were colonized by AM fungi, was similar in floodplain and higher elevation sites. It was as similar under ectomycorrhizal oaks as it was under red maples and other AM tree species. It was also similar among sites with deciduous understory shrubs with arbuscular mycorrhizae (spicebush, Lindera benzoin) and those with evergreen vegetation with ericoid mycorrhizae (mountain laurel, Kalmia latifolia). Where spicebush was the dominant understory shrub, inoculum potential was greater under gaps in the canopy than within the understory. Survivorship of transplanted red maple seedlings varied significantly over sites but was not strongly correlated with measures of inoculum potential. In a greenhouse growth experiment, arbuscular mycorrhizal fungal communities obtained from tree roots from the forest had different effects on plant growth. Seedlings inoculated with roots of red maple had twice the leaf area after 10 wk of growth compared to the AM community obtained from roots of southern red oaks. Thus, although there appears to be little heterogeneity in inoculum potential in the forest, there are differences in the effectiveness of different inocula. These effects have the potential to affect tree species diversity in forests by modifying patterns of seedling recruitment.

Transcriptome diversity among rice root types during asymbiosis and interaction with arbuscular mycorrhizal fungi.

Root systems consist of different root types (RTs) with distinct developmental and functional characteristics. RTs may be individually reprogrammed in response to their microenvironment to maximize adaptive plasticity. Molecular understanding of such specific remodeling-although crucial for crop improvement-is limited. Here, RT-specific transcriptomes of adult rice crown, large and fine lateral roots were assessed, revealing molecular evidence for functional diversity among individual RTs. Of the three rice RTs, crown roots displayed a significant enrichment of transcripts associated with phytohormones and secondary cell wall (SCW) metabolism, whereas lateral RTs showed a greater accumulation of transcripts related to mineral transport. In nature, arbuscular mycorrhizal (AM) symbiosis represents the default state of most root systems and is known to modify root system architecture. Rice RTs become heterogeneously colonized by AM fungi, with large laterals preferentially entering into the association. However, RT-specific transcriptional responses to AM symbiosis were quantitatively most pronounced for crown roots despite their modest physical engagement in the interaction. Furthermore, colonized crown roots adopted an expression profile more related to mycorrhizal large lateral than to noncolonized crown roots, suggesting a fundamental reprogramming of crown root character. Among these changes, a significant reduction in SCW transcripts was observed that was correlated with an alteration of SCW composition as determined by mass spectrometry. The combined change in SCW, hormone- and transport-related transcript profiles across the RTs indicates a previously overlooked switch of functional relationships among RTs during AM symbiosis, with a potential impact on root system architecture and functioning.

Responses of sugarcane, maize, and soybean to phosphorus and vesicular-arbuscular mycorrhizal fungi

The presence of vesicular-arbuscular mycorrhizal (VAM) fungi in long-term cane-growing fields associated with yield decline led to the supposition that VAM fungi may be responsible for the poor yields. A glasshouse trial was established to test the effectiveness of a species of VAM fungi, Glomus clarum, extracted from one of these North Queensland fields on the growth of sugarcane (Saccharum interspecific hybrid), maize (Zea mays), and soybean (Glycine max) for 6 phosphorus (P) rates (0, 2.7, 8.2, 25, 74, 222 mg/kg). For maize and soybean plants that received VAM (+ VAM), root colonisation was associated with enhanced P uptake, improved dry weight (DW) production, and higher index tissue-P concentrations than those without VAM (-VAM). By comparing DW responses of maize and soybean for different P rates, savings in fertiliser P of up to 160 and 213 kg/ha, respectively, were realised. Sugarcane plants were generally less responsive. Apart from a 30% DW increase with VAM when 2.7 mg P/kg was added, DW of +VAM plants was equivalent to, or worse than in the case of 222 mg P/kg, DW of -VAM plants. For all 3 host species, colonisation was least at the highest P application, presumably from excessive P within the plant tissue. Critical P concentrations for the 3 host species were below those reported elsewhere, and for soybean and sugarcane, the critical concentration for +VAM plants was lower than that of -VAM plants. There are 3 implications that arise from this study. First, VAM fungi present in cane-growing soils can promote the growth of maize and soybean, which are potential rotation crops, over a range of P levels. Second, the mycorrhizal strain taken from this site did not generally contribute to a yield decline in sugarcane plants. Third, application of P fertiliser is not necessary for sugarcane when acid-extractable P is

Molecular players shaping the arbuscular mycorrhizal symbiosis of rice

SUMMARY : The arbuscular mycorrhizal (AM) symbiosis is an evolutionarily ancient association between most land plants and Glomeromycotan fungi that is based on the mutual exchange of nutrients between the two partners. Its structural and physiological establishment is a multi-step process involving a tightly regulated signal exchange leading to intracellular colonization of roots by the fungi. Most research on the molecular biology and genetics of symbiosis development has been performed in dicotyledonous model legumes. In these, a plant signaling pathway, the common SYM pathway, has been found to be required for accommodation of both root symbionts rhizobia and AM fungi. Rice, a monocotyledon model and the world's most important staple crop also forms AM symbioses, has been largely ignored for studies of the AM symbiosis. Therefore in this PhD work functional conservation of the common SYM pathway in rice was addressed and demonstrated. Mycorrhiza-specific marker genes were established that are expressed at different stages of AM development and therefore represent readouts for various AM-specific signaling events. These tools were successfully used to obtain evidence for a yet unknown signaling network comprising common SYM-dependent and -independent events. In legumes AM colonization induces common SYM signaling dependent changes in root system architecture. It was demonstrated that also in rice, root system architecture changes in response to AM colonization but these alterations occur independently of common SYM signaling. The rice root system is complex and contains three different root types. It was shown that root type identity influences the quantity of AM colonization, indicating root type specific symbiotic properties. Interestingly, the root types differed in their transcriptional responses to AM colonization and the less colonized root type responded more dramatically than the more strongly colonized root type. Finally, in an independent project a novel mutant, inhospitable (iho), was discovered. It is perturbed at the most early step of AM colonization, namely differentiation of the AM fungal hyphae into a hyphopodium at the root surface. As plant factors required for this early step are not known, identification of the IHO gene will greatly contribute to the advance of mycorrhiza RÉSUMÉ : La symbiose mycorhizienne arbusculaire (AM) est une association évolutionnairement ancienne entre la majorité des plantes terrestres et les champignons du type Glomeromycota, basée sur l'échange mutuel d'éléments nutritifs entre les deux partenaires. Son établissement structural et physiologique est un processus en plusieurs étapes, impliquant des échanges de signaux étroitement contrôlés, aboutissant à la colonisation intracellulaire des racines par le champignon. La plupart des recherches sur la biologie moléculaire et la génétique du développement de la symbiose ont été effectuées sur des légumineuses dicotylédones modèles. Dans ces dernières, une voie de signalisation, la voie SYM, s'est avérée nécessaire pour permettre la mise en place de la symbiose mycorhizienne. Chez les plantes monocotylédones, comme le riz, une des céréales les plus importantes, nourrissant la moitié de la population mondiale, peu de recherches ont été effectuées sur les bases de la cette symbiose. Dans ce travail de thèse, la conservation fonctionnelle de la voie commune SYM chez le riz a été étudiée et démontrée. De plus, des gènes marqueurs spécifiques des différentes étapes du développement de l'AM ont été identifiés, permettant ainsi d'avoir des traceurs de la colonisation. Ces outils ont été utilisés avec succès pour démontrer l'existence d'un nouveau réseau de signalisation, comprenant des éléments SYM dépendant et indépendant. Chez les légumineuses, la colonisation par les AM induit des changements dans l'architecture du système racinaire, via la signalisation SYM dépendantes. Cependant chez le riz, il a été démontré que l'architecture de système racinaire changeait suite à la colonisation de l'AM, mais ceux, de façon SYM indépendante. Le système racinaire du riz est complexe et contient trois types différents de racines. Il a été démontré que le type de racine pouvait influencer l'efficacité de la colonisation par l'AM, indiquant que les racines ont des propriétés symbiotiques spécifiques différentes. De façon surprenante, les divers types de racines répondent de différemment suite à colonisation par l'AM avec des changements de la expression des gènes. Le type de racine le moins colonisé, répondant le plus fortement a la colonisation, et inversement. En parallèle, dans un projet indépendant, un nouveau mutant, inhospitable (iho), a été identifié. Ce mutant est perturbé lors de l'étape la plus précoce de la colonisation par l'AM, à savoir la différentiation des hyphes fongiques de l'AM en hyphopodium, à la surface des racines. Les facteurs d'origine végétale requis pour cette étape étant encore inconnus, l'identification du gène IHO contribuera considérablement a accroître nos connaissance sur les bases de la mise en place de cette symbiose.

Ecto- and arbuscular mycorrhizal symbiosis can induce tolerance to toxic pulses of phosphorus in jarrah (Eucalyptus marginata) seedlings

In common with many plants native to low P soils, jarrah (Eucalyptus marginata) develops toxicity symptoms upon exposure to elevated phosphorus (P). Jarrah plants can establish arbuscular mycorrhizal (AM) and ectomycorrhizal (ECM) associations, along with a non-colonizing symbiosis described recently. AM colonization is known to influence the pattern of expression of genes required for P uptake of host plants and our aim was to investigate this phenomenon in relation to P sensitivity. Therefore, we examined the effect on hosts of the presence of AM and ECM fungi in combination with toxic pulses of P and assessed possible correlations between the induced tolerance and the shoot P concentration. The P transport dynamics of AM (Rhizophagus irregularis and Scutellospora calospora), ECM (Scleroderma sp.), non-colonizing symbiosis (Austroboletus occidentalis), dual mycorrhizal (R. irregularis and Scleroderma sp.), and non-mycorrhizal (NM) seedlings were monitored following two pulses of P. The ECM and A. occidentalis associations significantly enhanced the shoot P content of jarrah plants growing under P-deficient conditions. In addition, S. calospora, A. occidentalis, and Scleroderma sp. all stimulated plant growth significantly. All inoculated plants had significantly lower phytotoxicity symptoms compared to NM controls 7 days after addition of an elevated P dose (30 mg P kg−1 soil). Following exposure to toxicity-inducing levels of P, the shoot P concentration was significantly lower in R. irregularis-inoculated and dually inoculated plants compared to NM controls. Although all inoculated plants had reduced toxicity symptoms and there was a positive linear relationship between rank and shoot P concentration, the protective effect was not necessarily explained by the type of fungal association or the extent of mycorrhizal colonization.

Nonredundant Regulation of Rice Arbuscular Mycorrhizal Symbiosis by Two Members of the PHOSPHATE TRANSPORTER1 Gene Family.

Pi acquisition of crops via arbuscular mycorrhizal (AM) symbiosis is becoming increasingly important due to limited high-grade rock Pi reserves and a demand for environmentally sustainable agriculture. Here, we show that 70% of the overall Pi acquired by rice (Oryza sativa) is delivered via the symbiotic route. To better understand this pathway, we combined genetic, molecular, and physiological approaches to determine the specific functions of two symbiosis-specific members of the PHOSPHATE TRANSPORTER1 (PHT1) gene family from rice, ORYsa;PHT1;11 (PT11) and ORYsa;PHT1;13 (PT13). The PT11 lineage of proteins from mono- and dicotyledons is most closely related to homologs from the ancient moss, indicating an early evolutionary origin. By contrast, PT13 arose in the Poaceae, suggesting that grasses acquired a particular strategy for the acquisition of symbiotic Pi. Surprisingly, mutations in either PT11 or PT13 affected the development of the symbiosis, demonstrating that both genes are important for AM symbiosis. For symbiotic Pi uptake, however, only PT11 is necessary and sufficient. Consequently, our results demonstrate that mycorrhizal rice depends on the AM symbiosis to satisfy its Pi demands, which is mediated by a single functional Pi transporter, PT11.

A novel bioinformatics pipeline to discover genes related to arbuscular mycorrhizal symbiosis based on their evolutionary conservation pattern among higher plants.

BACKGROUND: Genes involved in arbuscular mycorrhizal (AM) symbiosis have been identified primarily by mutant screens, followed by identification of the mutated genes (forward genetics). In addition, a number of AM-related genes has been identified by their AM-related expression patterns, and their function has subsequently been elucidated by knock-down or knock-out approaches (reverse genetics). However, genes that are members of functionally redundant gene families, or genes that have a vital function and therefore result in lethal mutant phenotypes, are difficult to identify. If such genes are constitutively expressed and therefore escape differential expression analyses, they remain elusive. The goal of this study was to systematically search for AM-related genes with a bioinformatics strategy that is insensitive to these problems. The central element of our approach is based on the fact that many AM-related genes are conserved only among AM-competent species. RESULTS: Our approach involves genome-wide comparisons at the proteome level of AM-competent host species with non-mycorrhizal species. Using a clustering method we first established orthologous/paralogous relationships and subsequently identified protein clusters that contain members only of the AM-competent species. Proteins of these clusters were then analyzed in an extended set of 16 plant species and ranked based on their relatedness among AM-competent monocot and dicot species, relative to non-mycorrhizal species. In addition, we combined the information on the protein-coding sequence with gene expression data and with promoter analysis. As a result we present a list of yet uncharacterized proteins that show a strongly AM-related pattern of sequence conservation, indicating that the respective genes may have been under selection for a function in AM. Among the top candidates are three genes that encode a small family of similar receptor-like kinases that are related to the S-locus receptor kinases involved in sporophytic self-incompatibility. CONCLUSIONS: We present a new systematic strategy of gene discovery based on conservation of the protein-coding sequence that complements classical forward and reverse genetics. This strategy can be applied to diverse other biological phenomena if species with established genome sequences fall into distinguished groups that differ in a defined functional trait of interest.

The half-size ABC transporters STR1 and STR2 are indispensable for mycorrhizal arbuscule formation in rice.

The central structure of the symbiotic association between plants and arbuscular mycorrhizal (AM) fungi is the fungal arbuscule that delivers minerals to the plant. Our earlier transcriptome analyses identified two half-size ABCG transporters that displayed enhanced mRNA levels in mycorrhizal roots. We now show specific transcript accumulation in arbusculated cells of both genes during symbiosis. Presently, arbuscule-relevant factors from monocotyledons have not been reported. Mutation of either of the Oryza sativa (rice) ABCG transporters blocked arbuscule growth of different AM fungi at a small and stunted stage, recapitulating the phenotype of Medicago truncatula stunted arbuscule 1 and 2 (str1 and str2) mutants that are deficient in homologous ABCG genes. This phenotypic resemblance and phylogenetic analysis suggest functional conservation of STR1 and STR2 across the angiosperms. Malnutrition of the fungus underlying limited arbuscular growth was excluded by the absence of complementation of the str1 phenotype by wild-type nurse plants. Furthermore, plant AM signaling was found to be intact, as arbuscule-induced marker transcript accumulation was not affected in str1 mutants. Strigolactones have previously been hypothesized to operate as intracellular hyphal branching signals and possible substrates of STR1 and STR2. However, full arbuscule development in the strigolactone biosynthesis mutants d10 and d17 suggested strigolactones to be unlikely substrates of STR1/STR2. Interestingly, rice STR1 is associated with a cis-natural antisense transcript (antiSTR1). Analogous to STR1 and STR2, at the root cortex level, the antiSTR1 transcript is specifically detected in arbusculated cells, suggesting unexpected modes of STR1 regulation in rice.

Ecological specificity of arbuscular mycorrhizae: evidence from foliar- and seed-feeding insects

Arbuscular mycorrhizal (AM) fungi have a variety of effects on foliar-feeding insects, with the majority of these being positive, although reports of negative and null effects also exist. Virtually all previous experiments have used mobile insects confined in cages and have studied the effects of one, or at most two, species of mycorrhizae on one species of insect. The purpose of this study was to introduce a greater level of realism into insect-mycorrhizal experiments, by studying the responses of different insect feeding guilds to a variety of AM fungi. We conducted two experiments involving three species of relatively immobile insects (a leaf-mining and two seed-feeding flies) reared in natural conditions on a host (Leucanthemum vulgare). In a field study, natural levels of AM colonization were reduced, while in a phytometer trial, we experimentally colonized host plants with all possible combinations of three known mycorrhizal associates of L. vulgare. In general, AM fungi increased the stature (height and leaf number) and nitrogen content of plants. However, these effects changed through the season and were,dependent on the identity of the fungi in the root system. AM fungi increased host acceptance of all three insects and larval performance of the leaf miner, but these effects were also season- and AM species-dependent. We suggest that the mycorrhizal effect on the performance of the leaf miner is due to fungal-induced changes in host-plant nitrogen content, detected by the adult fly. However, variability in the effect was apparent, because not all AM species increased plant N content. Meanwhile, positive effects of mycorrhizae were found on flower number and flower size, and these appeared to result in enhanced infestation levels by the seed-feeding insects. The results show that AM fungi exhibit ecological specificity, in that different. species have different effects on host-plant growth and chemistry and the performance of foliar-feeding insects. Future studies need to conduct experiments that use ecologically realistic combinations of plants and fungi and allow insects to be reared in natural conditions.

The diversity of arbuscular mycorrhizas of selected Australian Fabaceae

Members of the Australian native perennial Fabaceae have been little explored with regard to their root biology and the role played by arbuscular mycorrhizal (AM) fungi in their establishment, nutrition and long-term health. The ultimate goal of our research is to determine the dependency of native perennial legumes on their co-evolved AM fungi and conversely, the impact of AM fungal species in agricultural fields on the productivity of sown native perennial legume pastures. In this paper we investigate the colonisation morphology in roots and the AMF, identified by spores extracted from rhizosphere soil, from three replicate plots of each of the native legumes, Cullen australasicum, C. tenax and Lotus australis and the exotic legumes L. pedunculatus and Medicago sativa. The plants were grown in an agricultural field. The level and density of colonisation by AM fungi, and the frequency of intraradical and extraradical hyphae, arbuscules, intraradical spores and hyphal coils all differed between host plants and did not consistently differ between native and exotic species. However, there were strong similarities between species in the same genus. The three dominant species of AM fungi in rhizosphere soil also differed with host plant, but one fungus (Glomus mosseae) was always the most dominant. Sub-dominant AM species were the same between species in the same genus. No consistent differences in dominant spores were observed between the exotic and native Fabaceae species. Our results suggest that plant host influences the mycorrhizal community in the rhizosphere soil and that structural and functional differences in the symbiosis may occur at the plant genus level, not the species level or due to provenance.

Growth responses of sugarcane to mycorrhizal spore density and phosphorus rate

Arbuscular mycorrhizal (AM) fungi, commonly found in long-term cane-growing fields in northern Queensland, are linked with both negative and positive growth responses by sugarcane ( Saccharum spp.), depending on P supply. A glasshouse trial was established to examine whether AM density might also have an important influence on these growth responses. Mycorrhizal spores ( Glomus clarum), isolated from a long-term cane block in northern Queensland, were introduced into a pasteurised low-P cane soil at 5 densities ( 0, 0.06, 0.25, 1, 4 spores/g soil) and with 4 P treatments ( 0, 8.2, 25, and 47 mg/kg). At 83 days after planting, sugarcane tops responded positively to P fertilizer, although responses attributable to spore density were rarely observed. In one case, addition of 4 spores/g led to a 53% yield response over those without AM at 8 mg P/kg, or a relative benefit of 17 mg P/kg. Root colonisation was reduced for plants with nil or 74 mg P/kg. For those without AM, P concentration in the topmost visible dewlap ( TVD) leaf increased significantly with fertiliser P (0.07 v. 0.15%). However, P concentration increased further with the presence of AM spores. Irrespective of AM, the critical P concentration in the TVD leaf was 0.18%. This study confirms earlier reports that sugarcane is poorly responsive to AM. Spore density, up to 4 spores/g soil, appears unable to influence this responsiveness, either positively or negatively. Attempts to gain P benefits by increasing AM density through rotation seem unlikely to lead to yield increases by sugarcane. Conversely, sugarcane grown in fields with high spore densities and high plant-available P, such as long-termcane-growing soils, is unlikely to suffer a yield reduction from mycorrhizal fungi.