Characterisation of arbuscular mycorrhizal fungi colonisation in cluster roots of shape Hakea verrucosa F. Muell (Proteaceae), and its effect on growth and nutrient acquisition in ultramafic soil

Ultramafic soils at Bandalup Hill (Western Australia) are characterised by high concentrations of Ni and low levels of P. Amongst the plant species that can sustain such hostile conditions, Hakea verrucosa F. Muell from a non-mycorrhizal family (Proteaceae) would be expected to rely on cluster roots to access P. However, the acidification of ultramafic soils by cluster roots might increase the dissolution of soil Ni, and therefore its availability to plants. Symbiosis with mycorrhizal fungi, on the other hand, might help to reduce the uptake of Ni by H. verrucosa. Therefore, the aim of this study was to investigate the mycorrhizal status of H. verrucosa, and assess any contribution from mycorrhizal fungi to its growth and nutrient status. Seedlings of H. verrucosa were first grown in undisturbed ultramafic soil cores from Bandalup Hill for 8 weeks to assess the presence of mycorrhizal fungi in their roots. In a second experiment, H. verrucosa seedlings were grown in the same ultramafic soil that was either steamed or left untreated. Seedlings were inoculated with an arbuscular mycorrhizal (AM) fungal consortium from Bandalup Hill. Fungal hyphae, vesicles, as well as intracellular arbuscules and hyphal coils were observed in the cluster roots of H. verrucosa in both experiments. In the first experiment, 57% of the root length was colonized by AM fungi. Seedlings had high (between 1.4 and 1.9) shoot to root ratios and their roots had very few root hairs, despite growing in P-deficient soil. Steaming of the ultramafic soil increased the growth of seedlings and their nutrient uptake. Inoculation with AM fungi reduced the seedling growth in steamed ultramafic soil; however, it increased their shoot P and K concentration and also the shoot K content. The shoot Ni concentration of seedlings was not affected by the presence of AM fungi.     

Deficit and excess of soil water impact on plant growth of Lotus tenuis by affecting nutrient uptake and arbuscular mycorrhizal symbiosis

The impact of deficit and excess of soil water on plant growth, morphological plant features, N and P plant nutrition, soil properties, Rhizobium nodulation and the symbiosis between arbuscular mycorrhizal (AM) fungi and Lotus tenuis Waldst. & Kit. were studied in a saline-sodic soil. Water excess treatment decreased root growth by 36% and increased shoot growth by 13% whereas water deficit treatment decreased both root and shoot growth (26 and 32%, respectively). Differences between stress conditions on shoot growth were due to the ability of L. tenuis to tolerate low oxygen concentration in the soil and the sufficiency of nutrients in soil to sustain shoot growth demands. Water excess treatment decreased pH, and increased available P and labile C in soil. Water deficit treatment decreased available P and also increased labile C. In general, N and P acquisition were affected more by water excess than water deficit. The number of nodules per gram of fresh roots only increased in water excess roots (97%). Under both stress conditions there was a significant proportion of roots colonized by AM fungi. Compared to control treatment, arbuscule formation decreased by 55 and 14% under water excess and water deficit, respectively. Vesicle formation increased 256% in water excess treatment and did not change under water deficit treatment. L. tenuis plants subjected to water deficit or excess treatments could grow, nodulated and maintained a symbiotic association with AM fungi by different strategies. Under water excess, L. tenuis plants decreased root growth and increased shoot growth to facilitate water elimination by transpiration. Under water deficit, L. tenuis plants decreased root growth but also shoot growth which in turn significant decreased the shoot/root ratio. In the present study, under water excess conditions AM fungi reduced nutrient transfer structures (arbuscules), the number of entry points and spore, and hyphal densities in soil, but increased resistance structures (vesicles). At water deficit, however, AM fungi reduced external hyphae and arbuscules to some extent, investing more in maintaining a similar proportion of vesicles in roots and spores in soil compared to control treatment.     

Improved growth of salinity-stressed soybean after inoculation with salt pre-treated mycorrhizal fungi

Two sets of experiments to determine the effect of mycorrhiza on soybean (Glycine max) growth under saline conditions and to investigate the salt acclimation of mycorrhizal fungi were conducted. In the first experiment, the effect of an arbuscular mycorrhizal (AM) fungus Glomus etunicatum on mineral nutrient, proline and carbohydrate concentrations and growth of soybean. Under different NaCl concentrations (0, 50, 100, 150 and 200mM) was evaluated. Salinity decreased AM colonization. In both the M and nonAM plants shoot and root proline and shoot Na and Zn concentrations were increased under salinity. Soybean plants inoculated with the AM fungus had significantly higher fresh and dry weight, root proline, P, K and Zn but lower shoot proline and Na concentrations compared to the non inoculated plants. In the second experiment, the AM fungus was pre-treated with NaCl (salt acclimation) then was used as inoculum for soybean plants subjected to 100mM NaCl. Root colonization, fresh and dry weight, root proline, P, K and Zn concentrations were greater in soybean plants inoculated with the salt pre-treated fungus, compared to those inoculated with the nonsalt pre-treated fungus. However, for Na, the situation was the opposite. Based on these results, the AM inoculation helps the growth of soybean plants grown in saline conditions. When the AM fungus was pre-treated with NaCl with a gradual increase of concentration, and then exposed to a sudden salt stress, their efficiency was increased. This may be due to the acclimation of the AM fungus to salinity.     

Mycorrhiza-induced differential response to a yellows disease in tomato

The protective effects induced by arbuscular mycorrhizal (AM) fungi against a phytoplasma of the Stolbur group have been investigated in tomato by morphometry and flow cytometry. Symptoms induced by the phytoplasma were less severe when the plants also harboured AM fungi. Morphological parameters such as shoot and root fresh weight, shoot height, internode length, leaf number and adventitious root diameter were closer to those of healthy plants when arbuscular mycorrhiza were present. Reduced nuclear senescence was observed in AM plants infected with phytoplasmas; the percentages of nuclear populations with different ploidy levels were intermediate between AM and phytoplasma-infected plants. The mechanisms underlying these interactions are discussed and a direct action of the AM fungus is hypothesized.     

Some physiological responses of chickpea cultivars to arbuscular mycorrhiza under drought stress

A factorial experiment based on RCB design with three replicates was conducted to investigate changes in some physiological responses of two chickpea (Cicer arietinum L.) cultivars (Pirouz from Desi type and ILC482 from Kabuli type) to arbuscular mycorrhiza (Glomus etunicatum Becker and Gerdman) under different irrigation treatments. The experiment was carried out in the greenhouse of the Agricultural Faculty of Kurdistan University from April to August 2009. The results showed that leaf chlorophyll content of chickpea cultivars was significantly increased by arbuscular mycorrhiza (AM) under both well and limited irrigation conditions. Proline accumulation in chickpea leaves under moderate and severe drought stresses was significantly stronger than that under optimum irrigation. Inoculation of chickpea with mycorrhizal fungi caused an increase in the activities of polyphenol oxidase and peroxidase, but a decrease in the activity of catalase. Comparisons among different irrigation levels showed that chickpea plants under drought stress had the most active lipid peroxidation. Non-AM plants showed stronger lipid peroxidation under moderate and severe water stresses than AM plants. Lipid peroxidation was more active in Pirouz leaves than in ILC₄₈₂ leaves. It seems that Kabuli-type cultivar responded better to mycorrhizal symbiosis under drought stress than Desitype cultivar.     

Gibberellin regulates infection and colonization of host roots by arbuscular mycorrhizal fungi

Arbuscular mycorrhiza (AM) is established by the entry of AM fungi into the host plant roots and the formation of symbiotic structures called arbuscules. The host plant supplies photosynthetic products to the AM fungi, which in return provide phosphate and other minerals to the host through the arbuscules. Both partners gain great advantages from this symbiotic interaction, and both regulate AM development. Our recent work revealed that gibberellic acids (GAs) are required for AM development in the legume Lotus japonicus. GA signaling interact with symbiosis signaling pathways, directing AM fungal colonization in host roots. Expression analysis showed that genes for GA biosynthesis and metabolism were induced in host roots around AM fungal hyphae, suggesting that the GA signaling changes with both location and time during AM development. The fluctuating GA concentrations sometimes positively and sometimes negatively affect the expression of AM-induced genes that regulate AM fungal infection and colonization.     

Mycorrhizal dependency of banana (Musa acuminata,AAA group) cultivar

Seven banana cultivars (Musa acuminata, AAA group) were inoculated with two species of vesicular arbuscular mycorrhizal (VAM) fungi (Glomus mosseae and Glomus macrocarpum) in a greenhouse experiment. Inoculated plants had generally greater shoot dry weight and shoot phosphorus concentrations compared to the noninoculated plants. A great variation in dependency on mycorrhizal colonization was observed among the banana cultivars. Cv. Williams showed the highest relative mycorrhizal dependency (RMD) and cv. Poyo the lowest. For all the cultivars studied, inoculation with G. macrocarpum resulted in the highest RMD values. Both root dry weight and root hair length or density of the noninoculated plants were inverserly correlated with the RMD values of cultivars.     

Phosphorus,arbuscular mycorrhizal fungi and performance of the wetland plant Lythrum salicaria L. under inundated conditions

The role of arbuscular mycorrhizal (AM) fungi in aquatic and semi-aquatic environments is poorly understood, although they may play a significant role in the establishment and maintenance of wetland plant communities. We tested the hypothesis that AM fungi have little effect on plant response to phosphorus (P) supply in inundated soils as evidenced by an absence of increased plant performance in inoculated (AM+) versus non-inoculated (AM-) Lythrum salicaria plants grown under a range of P availabilities (0-40 mg/l P). We also assessed the relationship between P supply and levels of AM colonization under inundated conditions. The presence of AM fungi had no detectable benefit for any measures of plant performance (total shoot height, shoot dry weight, shoot fresh weight, root fresh weight, total root length or total root surface area). AM+ plants displayed reduced shoot height at 10 mg/l P. Overall, shoot fresh to dry weight ratios were higher in AM+ plants although the biological significance of this was not determined. AM colonization levels were significantly reduced at P concentrations of 5 mg/l and higher. The results support the hypothesis that AM fungi have little effect on plant response to P supply in inundated conditions and suggest that the AM association can become uncoupled at relatively high levels of P supply.     

Mycorrhizal fungal growth responds to soil characteristics,but not host plant identity,during a primary lacustrine dune succession

Soil factors and host plant identity can both affect the growth and functioning of mycorrhizal fungi. Both components change during primary succession, but it is unknown if their relative importance to mycorrhizas also changes. This research tested how soil type and host plant differences among primary successional stages determine the growth and plant effects of arbuscular mycorrhizal (AM) fungal communities. Mycorrhizal fungal community, plant identity, and soil conditions were manipulated among three stages of a lacustrine sand dune successional series in a fully factorial greenhouse experiment. Late succession AM fungi produced more arbuscules and soil hyphae when grown in late succession soils, although the community was from the same narrow phylogenetic group as those in intermediate succession. AM fungal growth did not differ between host species, and plant growth was similarly unaffected by different AM fungal communities. These results indicate that though ecological filtering and/or adaptation of AM fungi occurs during this primary dune succession, it more strongly reflects matching between fungi and soils, rather than interactions between fungi and host plants. Thus, AM fungal performance during this succession may not depend directly on the sequence of plant community succession.     

Linking lipid transfer with reduced arbuscule formation in tomato roots colonized by arbuscular mycorrhizal fungus under low pH stress

Arbuscules are the core structures of arbuscular mycorrhizae (AM), and arbuscule development is regulated by environmental stress, e.g., low pH. Recent studies indicate that lipid transfer from plants is essential for AM fungal colonization; however, the role of lipid transfer in arbuscule formation and the dynamics of lipid accumulation in arbuscules under low pH stress are far from well understood. In the symbiosis of tomato and Rhizophagus intraradices under contrasting pH conditions (pH 4.5 vs. pH 6.5), we investigated arbuscule formation, nutrient uptake, alkaline phosphatase activity and lipid accumulation; examined the gene expression involved in phosphate transport, lipid biosynthesis and transfer and sugar metabolism; and visualized the lipid dynamics in arbuscules. Low pH greatly inhibited arbuscule formation, in parallel with reduced phospholipid fatty acids accumulation in AM fungus and decreased P uptake. This reduction was supported by the decreased expression of plant genes encoding lipid biosynthesis and transfer. More degenerating arbuscules were observed under low pH conditions, and neutral lipid fatty acids accumulated only in degenerating arbuscules. These data reveal that, under low pH stress, reduced lipid transfer from hosts to AM fungi is responsible for the inhibited arbuscule formation.     

Effects of co-inoculation with arbuscular mycorrhizal fungi and rhizobia on soybean growth as related to root architecture and availability of N and P

Soybean plants can form tripartite symbiotic associations with rhizobia and arbuscular mycorrhizal (AM) fungi, but little is known about effects of co-inoculation with rhizobia and AM fungi on plant growth, or their relationships to root architecture as well as nitrogen (N) and phosphorus (P) availability. In the present study, two soybean genotypes contrasting in root architecture were grown in a field experiment to evaluate relationships among soybean root architecture, AMF colonization, and nodulation under natural conditions. Additionally, a soil pot experiment in greenhouse was conducted to investigate the effects of co-inoculation with rhizobia and AM fungi on soybean growth, and uptake of N and P. Our results indicated that there was a complementary relationship between root architecture and AMF colonization in the field. The deep root soybean genotype had greater AMF colonization at low P, but better nodulation with high P supply than the shallow root genotype. A synergistic relationship dependent on N and P status exists between rhizobia and AM fungi on soybean growth. Co-inoculation with rhizobia and AM fungi significantly increased soybean growth under low P and/or low N conditions as indicated by increased shoot dry weight, along with plant N and P content. There were no significant effects of inoculation under adequate N and P conditions. Furthermore, the effects of co-inoculation were related to root architecture. The deep root genotype, HN112, benefited more from co-inoculation than the shallow root genotype, HN89. Our results elucidate new insights into the relationship between rhizobia, AM fungi, and plant growth under limitation of multiple nutrients, and thereby provides a theoretical basis for application of co-inoculation in field-grown soybean.     

Effect of antifungal genes expressed in transgenic pea (Pisum sativum L.) on root colonization with Glomus intraradices

Pathogenic fungi have always been a major problem in agriculture. One of the effective methods for controlling pathogen fungi to date is the introduction of resistance genes into the genome of crops. It is interesting to find out whether the induced resistance in crops will have a negative effect on non-target organisms such as root colonization with the AM fungi. The objective of the present research was to study the influence of producing antifungal molecules by four transgenic pea (Pisum sativum L.) lines expressing PGIP gene from raspberry, VST-stilbene synthase from vine, a hybrid of PGIP/VST and bacterial Chitinase gene (Chit30) from Streptomyces olivaceoviridis respectively on the colonization potential of Glomus intraradices. Four different experiments were done in greenhouse and climate chamber, colonization was observed in all replications. The following parameters were used for evaluation: frequency of mycorrhization, the intensity of mycorrhization, the average presence of arbuscules within the colonized areas and the presence of arbuscules in the whole root system which showed insignificant difference between transgenic and non-transgenic plants. The root/shoot ratio exhibited different values according to the experiment condition. Compared with negative non-transgenic control all transgenic lines showed the ability to establish symbiosis and the different growth parameters had insignificant effect due to mycorrhization. The results of the present study proved that the introduced pathogen resistance genes did not affect the mycorrhization allocations in pea.     

Sex-Related Responses of Populus cathayana Shoots and Roots to AM Fungi and Drought Stress

We investigated the impact of drought and arbuscular mycorrhizal (AM) fungi on the morphological structure and physiological function of shoots and roots of male and female seedlings of the dioecious plant Populus cathayana Rehder. Pot-grown seedlings were subjected to well watered or water-limiting conditions (drought) and were grown in soil that was either inoculated or not inoculated with the AM fungus Rhizophagus intraradices. No significant differences were found in the infection rates between the two sexes. Drought decreased root and shoot growth, biomass and root morphological characteristics, whereas superoxide radical (O₂–) and hydrogen peroxide content, peroxidase (POD) activity, malondialdehyde (MDA) concentration and proline content were significantly enhanced in both sexes. Male plants that formed an AM fungal symbiosis showed a significant increase in shoot and root morphological growth, increased proline content of leaves and roots, and increased POD activity in roots under both watering regimes; however, MDA concentration in the roots decreased. By contrast, AM fungi either had no effect or a slight negative effect on the shoot and root growth of female plants, with lower root biomass, total biomass and root/shoot ration under drought. In females, MDA concentration increased in leaves and roots under both watering regimes, and the proline content and POD activity of roots increased under drought conditions; however, POD activity significantly decreased under well-watered conditions. These findings suggest that AM fungi enhanced the tolerance of male plants to drought by improving shoot and root growth, biomass and the antioxidant system. Further investigation is needed to unravel the complex effects of AM fungi on the growth and antioxidant system of female plants.     

Two Lotus japonicus symbiosis mutants impaired at distinct steps of arbuscule development

Arbuscular mycorrhiza (AM) fungi form nutrient‐acquiring symbioses with the majority of higher plants. Nutrient exchange occurs via arbuscules, highly branched hyphal structures that are formed within root cortical cells. With a view to identifying host genes involved in AM development, we isolated Lotus japonicus AM‐defective mutants via a microscopic screen of an ethyl methanesulfonate‐mutagenized population. A standardized mapping procedure was developed that facilitated positioning of the defective loci on the genetic map of L. japonicus, and, in five cases, allowed identification of mutants of known symbiotic genes. Two additional mutants representing independent loci did not form mature arbuscules during symbiosis with two divergent AM fungal species, but exhibited signs of premature arbuscule arrest or senescence. Marker gene expression patterns indicated that the two mutants are affected in distinct steps of arbuscule development. Both mutants formed wild‐type‐like root nodules upon inoculation with Mesorhizobium loti, indicating that the mutated loci are essential during AM but not during root nodule symbiosis.     

Arbuscular Mycorrhizal Fungi Promote the Growth of Ceratocarpus arenarius (Chenopodiaceae) with No Enhancement of Phosphorus Nutrition

The mycorrhizal status of plants in the Chenopodiaceae is not well studied with a few controversial reports. This study examined arbuscular mycorrhizal (AM) colonization and growth response of Ceratocarpus arenarius in the field and a greenhouse inoculation trial. The colonization rate of AM fungi in C. arenarius in in-growth field cores was low (around 15%). Vesicles and intraradical hyphae were present during all growth stages, but no arbuscules were observed. Sequencing analysis of the large ribosomal rDNA subunit detected four culturable Glomus species, G. intraradices, G. mosseae, G. etunicatum and G. microaggregatum together with eight unculturable species belong to the Glomeromycota in the root system of C. arenarius collected from the field. These results establish the mycotrophic status of C. arenarius. Both in the field and in the greenhouse inoculation trial, the growth of C. arenarius was stimulated by the indigenous AM fungal community and the inoculated AM fungal isolates, respectively, but the P uptake and concentration of the mycorrhizal plants did not increase significantly over the controls in both experiments. Furthermore, the AM fungi significantly increased seed production. Our results suggest that an alternative reciprocal benefit to carbon-phosphorus trade-off between AM fungi and the chenopod plant might exist in the extremely arid environment.     

Effects of preplant inoculation of apple (Malus domestica Borkh.) with arbuscular mycorrhizal fungi on population growth of the root-lesion nematode,Pratylenchus penetrans

Six species of arbuscular mycorrhizal (AM) fungi (Glomus aggregatum, G. clarum, G. etunicatum, G. intraradices, G. mosseae and G. versiforme) were evaluated, in three greenhouse experiments, for their effects on reproduction of the root-lesion nematode, Pratylenchus penetrans, and growth of Ottawa 3 apple rootstock. Glomus mosseae increased total dry weights of nematode-inoculated and non-inoculated rootstock in all three greenhouse experiments, and G. intraradices increased dry weights in two of three greenhouse experiments. Plants inoculated with G. mosseae generally supported fewer P. penetrans per gram of root than plants inoculated with other AM fungi, but did not differ significantly from the controls in any greenhouse experiment. Colonization of roots by AM fungi was reduced by P. penetrans at initial inoculum densities greater than 250 nematodes/L soil. In field trials, preplant inoculation with either G. intraradices or G. mosseae increased rootstock growth and leaf concentrations of P, Mg, Zn and Cu in fumigated plots but not in non-fumigated plots, indicating that colonization by native AM fungi in non-fumigated plots may have been sufficient for adequate nutrient acquisition. The abundance of vesicles and arbuscules was greater in roots of plants inoculated with AM fungi before planting than in roots of non-inoculated plants, in both fumigated and non-fumigated plots. P. penetrans per gram of root and per 50 ml soil were significantly lower for G. mosseae- inoculated plants than for non-inoculated plants in fumigated soil but not in non-fumigated soil.     

Interactions between biochar and mycorrhizal fungi in a water-stressed agricultural soil

Biochar may alleviate plant water stress in association with arbuscular mycorrhizal (AM) fungi but research has not been conclusive. Therefore, a glasshouse experiment was conducted to understand how interactions between AM fungi and plants respond to biochar application under water-stressed conditions. A twin chamber pot system was used to determine whether a woody biochar increased root colonisation by a natural AM fungal population in a pasture soil (‘field’ chamber) and whether this was associated with increased growth of extraradical AM fungal hyphae detected by plants growing in an adjacent (‘bait’) chamber containing irradiated soil. The two chambers were separated by a mesh that excluded roots. Subterranean clover was grown with and without water stress and harvested after 35, 49 and 63 days from each chamber. When biochar was applied to the field chamber under water-stressed conditions, shoot mass increased in parallel with mycorrhizal colonisation, extraradical hyphal length and shoot phosphorus concentration. AM fungal colonisation of roots in the bait chamber indicated an increase in extraradical mycorrhizal hyphae in the field chamber. Biochar had little effect on AM fungi or plant growth under well-watered conditions. The biochar-induced increase in mycorrhizal colonisation was associated with increased growth of extraradical AM fungal hyphae in the pasture soil under water-stressed conditions.     

A purple acid phosphatase,GmPAP33, participates in arbuscule degeneration during arbuscular mycorrhizal symbiosis in soybean

Arbuscules are the central structures of the symbiotic association between terrestrial plants and arbuscular mycorrhizal (AM) fungi. However, arbuscules are also ephemeral structures, and following development, these structures are soon digested and ultimately disappear. Currently, little is known regarding the mechanism underlying the digestion of senescent arbuscules. Here, biochemical and functional analyses were integrated to test the hypothesis that a purple acid phosphatase, GmPAP33, controls the hydrolysis of phospholipids during arbuscule degeneration. The expression of GmPAP33 was enhanced by AM fungal inoculation independent of the P conditions in soybean roots. Promoter‐β‐glucuronidase (GUS) reporter assays revealed that the expression of GmPAP33 was mainly localized to arbuscule‐containing cells during symbiosis. The recombinant GmPAP33 exhibited high hydrolytic activity towards phospholipids, phosphatidylcholine, and phosphatidic acid. Furthermore, soybean plants overexpressing GmPAP33 exhibited increased percentages of large arbuscules and improved yield and P content compared with wild‐type plants when inoculated with AM fungi. Mycorrhizal RNAi plants had high phospholipid levels and a large percentage of small arbuscules. These results in combination with the subcellular localization of GmPAP33 at the plasma membrane indicate that GmPAP33 participates in arbuscule degeneration during AM symbiosis via involvement in phospholipid hydrolysis.     

Occurrence of arbuscular mycorrhiza in Castanospermum australe A. Cunn. & C. Fraser and effects on growth and production of castanospermine

 Castanospermum australe A. Cunn. & C. Fraser is the only species of the genus Castanospermum (the Moreton Bay chestnut or black bean) native to NE Australia. One constituent of the plant, castanospermine, can inhibit the AIDS virus. The present study investigated possible symbioses between its roots and arbuscular mycorrhizal (AM) fungi. The effects of mycorrhizal fungi on the growth of the plant and yield of alkaloid castanospermine were also studied. The mycorrhizosphere soil and roots of C. australe collected from various sites in and around Sydney, Australia showed AM symbiotic associations with roots, with arbuscules and vesicles in the root cortices. Wet sieving and decanting yielded AM fungal spores, mainly Glomus spp. A positive correlation was found between AM fungal infection and the castanospermine content of seeds of field-grown trees. Field study results were confirmed by growing seedlings under greenhouse conditions and inoculating them with Glomus intraradices Schenck and Smith (INVAM isolate KS906) and Gigaspora margarita Becker & Hall (INVAM isolate BR444–2). The AM fungi increased the growth and P contents of plants and the yield of castanospermine in the leaves, irrespective of the P treatment. No correlation was found between the alkaloid contents of leaves from mycorrhizal seedlings and from non-mycorrhizal plants which received P. No significant difference in the production of castanospermine was found between P treatments when G. margarita was used as inoculum. Accepted: 14 April 1999