Rationalizing molecular analysis of field-collected roots for assessing diversity of arbuscular mycorrhizal fungi: to pool, or not to pool, that is the question

For rationalizing molecular analysis of field-collected roots in diversity studies on arbuscular mycorrhiza, we compared three different approaches. After DNA extraction from 50 root samples of Plantago lanceolata grown on monoculture plots at a former arable field site, (1) DNAs were amplified separately by nested PCR and each amplicon was cloned separately; (2) DNAs were amplified separately by nested PCR, 1 μl of each amplicon was pooled, and a single cloning was made from the resulting amplicons mix; and (3) DNAs were pooled and the single amplicon derived from the nested PCR was cloned. Based on these three different methods, 109 nuclear ribosomal internal transcribed spacer sequences were obtained. Methods 1 and 2 enabled the detection of almost similar levels of arbuscular mycorrhizal fungal diversity. However, method 1 was expensive and time-consuming as much more cloning had to be done. Method 3 was completely biased by preferential amplification of nontarget organisms, which were only detected in low frequencies by the other methods.     

Differential effect of sample preservation methods on plant and arbuscular mycorrhizal fungal DNA

A wide range of methods are commonly used for preserving environmental samples prior to molecular analyses. However, the effect of these preservation methods on fungal DNA is not understood. The objective of this study was to test the effect of eight different preservation methods on the quality and yield of DNA extracted from Bromus inermis and Daucus carota roots colonized by the arbuscular mycorrhizal (AM) fungus, Glomus intraradices. The total DNA concentration in sample extracts was quantified using spectrophotometry. Samples that were frozen (− 80 ºC and − 20 ºC), stored in 95% ethanol, or silica gel dried yielded total (plant and fungal) DNA concentrations that were not significantly different from fresh samples. In contrast, samples stored in CTAB solution or freeze-dried resulted in significantly reduced DNA concentrations compared with fresh samples. The preservation methods had no effect on the purity of the sample extracts for both plant species. However, the DNA of the dried samples (silica gel dried, freeze-dried, heat dried) appeared to be slightly more degraded compared with samples that remained hydrated (frozen, stored in ethanol or CTAB solutions) during storage when visualized on a gel. The concentration of AM fungal DNA in sample extracts was quantified using TaqMan real time PCR. Methods that preserved samples in hydrated form had similar AM fungal DNA concentrations as fresh samples, except D. carota samples stored in ethanol. In contrast, preservation methods that involved drying the samples had very low concentrations of AM fungal DNA for B. inermis, and nearly undetectable for D. carota samples. The drying process appears to be a major factor in the degradation of AM fungal DNA while having less of an impact on plant DNA. Based on these results, samples that need to be preserved prior to molecular analysis of AM fungi should be kept frozen to minimize the degradation of plant and AM fungal DNA.     

Time-course study and partial characterization of a protein on hyphae of arbuscular mycorrhizal fungi during active colonization of roots

Material on the surface of hyphal walls of arbuscular mycorrhizal fungi (AMF) during active colonization of plant roots was detected by a monoclonal antibody. Pot-cultured isolates of Glomus, Acaulospora, Gigaspora, Scutellospora, and Entrophospora had immunofluorescent material (IM) on younger, thinner, intact hyphae, but IM was scant to absent on thicker, melanized or lysing hyphae. Colonization of corn (Zea mays L.), Sudangrass (Sorghum sudanense (Piper) Staph.) or red clover (Trifolium pratense L.) was examined during 5 months of plant growth by removing cores and performing an indirect immunoassay on roots with attached hyphae. Fresh spores of some Glomus spp. had IM on the outer layer of the spore wall. Abundant IM was seen on root hairs of plants colonized by some isolates, and some IM was detected on root surfaces of all plants examined even during early colonization. After cultures were dried, hyphae, roots and spores had little to no IM. Uninoculated control roots had very rare, small patches of IM. An immunoreactive protein was extracted from hyphae of Gigaspora and Glomus isolates by using 20mM citrate (pH 7.0) at 121°C for 90 min. Gel electrophoresis profiles indicated that all isolates tested had the same banding patterns. Lectin-binding of extracted protein is suggestive of a glycoprotein. The immunofluorescence assay can be used to examine root sections for active colonization by AMF, and the potential use of the protein to quantify AMF activity in soil is discussed.     

A simple and reliable method for SSU rRNA gene DNA extraction, amplification, and cloning from single AM fungal spores

 We describe a method that allows quick and easy PCR amplification and cloning of nearly complete SSU rRNA genes from arbuscular mycorrhizal fungi. The procedure tested on spores from 37 different glomalean isolates was based on magnetic separation with Dynabeads, followed by nested PCR with two primer pairs. All trials led to visible amplification products of the expected size. Thereafter, the PCR fragments could be quickly and efficiently cloned by means of a topoisomerase-activated vector (pCR2.1-TOPO). The technique is rapid, uncomplicated and comparatively inexpensive. The use of single spores for DNA extraction has some advantages over multispore-preparations, e.g. it is less susceptible to contamination with other organisms present in the cultures. The method can be used for the quick and reliable preparation of a large number of samples and is highly reproducible. It could also be used for genes other than the SSU rRNA gene. Accepted: 25 October 2000     

柑橘丛枝菌根真菌生长与根际有效磷和磷酸酶活性的相关性

通过观测田间国庆1号温州密柑/枳和国庆4号温州密柑/枳根系菌根侵染率、孢子密度、根际有效磷和磷酸酶活性的年变化,探讨丛枝菌根真菌生长与根际有效磷和磷酸酶活性的相关性.结果表明,2种柑橘菌根侵染率和孢子密度的年变化均呈 “Λ”形,2月和12月较低,4月和10月居中,6月和8月较高;有效磷和中性磷酸酶年变化呈“V”形.2种柑橘的菌根侵染率都与孢子密度呈极显著正相关,与有效磷呈极显著负相关,说明较高的孢子密度和较低的有效磷对菌根侵染率有促进作用;2种柑橘的孢子密度均与有效磷呈极显著负相关,与中性磷酸酶和总磷酸酶呈极显著正相关,表明中性磷酸酶和总磷酸酶对孢子密度有刺激作用,而有效磷对其有抑制作用.柑橘树下有机磷矿化主要以中性磷酸酶为主.     

丛枝菌根观察与侵染率测定方法的比较

菌根生长状况观察与侵染率测定是菌根学研究中一项重要的基础性工作。综述了丛枝菌根（AM）染色观察与侵染率测定方法研究概况,并对其进行比较和评价。认为采用醋酸墨水染色观察AM生长状况与采用根段侵染率加权法和放大交叉法测定AM真菌侵染率是目前较为科学、准确、易行的方法。根据不同需要也可选择其他适宜的方法,如要了解丛枝发育状况,可采用放大交叉法;如要了解泡囊和侵入点数量,可采用直接计数法,从而使其研究结果具有可比性。有必要建立基于分子生物学技术和脂肪酸定量分析技术测定一种或数种AM真菌侵染状况,这将有力推动AM真菌生理、生态功能研究的发展。     

中国大陆地区丛枝菌根真菌菌种资源的分离鉴定与形态学特征

【目的】分离收集保藏中国大陆各个地区不同生态环境的丛枝菌根真菌菌种资源,为丛枝菌根的研究提供资源、奠定基础。【方法】以高粱为宿主植物,采用诱导培养、单孢培养和扩繁培养分离土壤样品中的丛枝菌根真菌菌种并鉴定。【结果】从我国大陆的45个地区50余种宿主植物根区土壤中分离到丛枝菌根真菌135株,隶属于23个种;对各个菌株的形态特征进行了描述。【结论】我国蕴藏着丰富的丛枝菌根真菌菌种资源,文中描述的菌种资源是目前从我国大陆地区获得的种类和数量最多、覆盖范围最广的AM真菌菌种资源。     

4种黑曲霉基因组DNA提取方法的比较

目的:筛选适合提取曲霉DNA的方法.方法:比较2个菌落培养时间段(3d内和10d左右)提取DNA质量的差异;运用氯化苄法、石英砂+CTAB法、Biospin法和微波法分别提取黑曲霉基因组DNA,然后用直接电泳、浓度测定、PCR扩增等方法比较所提DNA的浓度和质量.结果:培养3d内的菌落提取的DNA纯度较高,无需纯化即可用于后续实验;4种方法制备的DNA均可用于PCR等后续实验,其中以石英砂+CTAB法提取的DNA纯度好,产率最高.结论石英砂+CTAB法是一种适用于曲霉DNA提取的简便方法.     

Rapid and efficient extraction of genomic DNA from differentphytopathogenic fungi using DNAzol reagent

A modified procedure using the commercial DNAzol reagent was successfully applied to extract genomic DNA from 25 fungal species. The DNA yield varied from 306 to 1,927 g g^-1 dry mycelia and the A₂₆₀/A₂₈₀ ratio from 1.59 to 1.93. Compared with the method of J.L. Cenis (Nucleic Acids Res. 1992, 20: 2380) this procedure generated a higher DNA yield from 17 species and a higher A₂₆₀/A₂₈₀ ratio from 23 species. But for four species, Cenis (1992) method was more suitable. No inhibitor of polymerase chain reaction was evident for the DNA extracted by the modified procedure, whereas some inhibitors remained in DNA of eight species extracted by the previous method.Revisions requested 8 September 2004; Revisions received 1 November 2004     

Comparing symbiotic performance and physiological responses of two soybean cultivars to arbuscular mycorrhizal fungi under salt stress

The presented experiments evaluated the symbiotic performance of soybean genotypes with contrasting salt stress tolerance to arbuscular mycorrhizal fungi (AMF) inoculation. In addition, the physiological stress tolerance mechanisms in plants derived from mutualistic interactions between AMF and the host plants were evaluated. Plant growth, nodulation, nitrogenase activity and levels of endogenous growth hormones, such as indole acetic acid and indole butyric acid, of salt-tolerant and salt-sensitive soybean genotypes significantly decreased at 200 mM NaCl. The inoculation of soybean with AMF improved the symbiotic performance of both soybean genotypes by improving nodule formation, leghemoglobin content, nitrogenase activity and auxin synthesis. AMF colonization also protected soybean genotypes from salt-induced membrane damage and reduced the production of hydrogen peroxide, subsequently reducing the production of TBARS and reducing lipid peroxidation. In conclusion, the results of the present investigation indicate that AMF improve the symbiotic performance of soybean genotypes regardless of their salt stress tolerance ability by mitigating the negative effect of salt stress and stimulating endogenous level of auxins that contribute to an improved root system and nutrient acquisition under salt stress.     

Diversity of Cryptococcus and Dioszegia yeasts (Basidiomycota) inhabiting arbuscular mycorrhizal roots or spores

The genera Cryptococcus and Dioszegia contain basidiomycetous yeasts found in a wide range of habitats. Primers to amplify the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA (nrDNA) of arbuscular mycorrhizal fungi (AMF) also allow detecting members of this yeast group. Here we report the results of a sequence analysis using maximum parsimony on a set of 50 ITS sequences of yeasts associated with AMF structures (roots of 26 plant species, AM spores) from six field sites in Central Germany. Among 10 separated taxa, respectively five in the Tremellales and two in the Filobasidiales had unknown sequences. Therefore it was not possible to assign these sequences to any known species. The study indicates that exploring the diversity of Cryptococcus and Dioszegia in soil habitats with molecular methods might enlarge the actually estimated biodiversity of the group.     

Optimisation of DNA extraction for AFLP analysis of mycorrhizal fungi of terrestrial orchids caladeniinae and drakaeinae

Published DNA extraction methods present a number of problems when applied to mycorrhizal fungi of native Australian terrestrial orchids. Grinding with liquid nitrogen shears the DNA, and other pulverisation methods yield too little DNA. We found that freezing the fungal sample with liquid nitrogen, with no grinding, followed by the Qiagen DNeasy extraction procedure produced good yields of high-molecular-weight DNA. The DNA was then used for amplified fragment length polymorphism (AFLP) fingerprinting. Good fingerprints were produced by restriction withEcoRI/MseI enzymes, the use of preamplification primer mix II (for small genomes), and a 2-base extensionMseI primer (m-cc) with 3-base extensionEcoRI primers in the selective amplification. This protocol may be of general utility for other fungi with similarly fragile DNA.     

Arbuscular mycorrhizal fungi in the tree seedlings of two Australian rain forests: occurrence, colonization, and relationships with plant performance

The roots of rain forest plants are frequently colonized by arbuscular mycorrhizal fungi (AMF) that can promote plant growth in the nutrient poor soils characteristic of these forests. However, recent studies suggest that both the occurrence of AMF on rain forest plants and the dependence of rain forest plants on AMF can be highly variable. We examined the occurrence and levels of AMF colonization of some common seedling species in a tropical and a subtropical rain forest site in Queensland, Australia. We also used a long-term database to compare the growth and mortality rates of seedling species that rarely formed AMF with those that regularly formed AMF. In both forests, more than one-third of the seedling species rarely formed AMF associations, while 40% of species consistently formed AMF in the tropical site compared to 27% in the subtropical site. Consistent patterns of AMF occurrence were observed among plant families at the two sites. Variation among seedling species in AMF occurrence or colonization was not associated with differences in seed mass among species, variation in seedling size and putative age within a species, or lack of AMF inoculum in the soil. Comparisons of four seedling species growing both in the shaded understory and in small canopy gaps revealed an increase in AMF colonization in two of the four species in gaps, suggesting that light limitation partially explains the low occurrence of AMF. Seedling survival was significantly positively associated with seed biomass but not with AMF colonization. Furthermore, seedling species that regularly formed AMF and those that did not had similar rates of growth and survival, suggesting that mycorrhizal and nonmycorrhizal strategies were equivalent in these forests. Furthermore, the high numbers of seedlings that lacked AMF and the overall low rate of seedling growth (the average seedling required 6 years to double its height) suggest that most seedlings did not receive significant indirect benefits from AMF through connection to canopy trees via a common mycorrhizal network.     

Comparison of two test systems for measuring plant phosphorus uptake via arbuscular mycorrhizal fungi

 Plant phosphorus uptake via external hyphae of arbuscular mycorrhizal fungi has been measured using compartmented systems where a hyphal compartment is separated from a rooting compartment by a fine mesh. By labelling the soil within the hyphal compartment with a radioactive phosphorus (P) isotope, hyphal uptake of P into the plant can be traced. The objective of this growth chamber study was to test two hyphal compartments of different design with respect to their suitabilities for measurement of hyphal P uptake. One hyphal compartment was simply a nylon mesh bag filled with ³²P-labelled soil. The labelled soil in the other hyphal compartment was completely surrounded by an 8–10 mm layer of unlabelled soil that served as a buffer zone. Mycorrhizal and non-mycorrhizal subterranean clover plants were grown in pots with a centrally positioned hyphal compartment. Uptake of radioactive P by non-mycorrhizal control plants was 25% of that by mycorrhizal plants with the mesh bag but only 3% when including the buffer zone. Based on this good control of non-mycorrhizal P uptake from within the hyphal compartment and its greater ease of handling once produced, we judged the hyphal compartment including a buffer zone to be superior to the mesh bag. Accepted: 15 September 1998     

一种适用于PCR反应的酵母菌、无绿藻及丝状真菌DNA提取方法

目的 介绍一种从酵母、无绿藻及丝状真菌中提取DNA以用于PCR反应的方法。方法 所用菌种包括临床分离的未知菌株和保藏菌株共23株：未知酵母菌（5株）、真皮毛孢子菌（1株）、糠秕马拉色菌（1株）、季也蒙念珠菌（5株）、未知丝状真菌（6株）、无绿藻（1株）、烟曲霉（2株）、拟青霉菌（1株）、茎点霉（1株）。用溶细胞酶（lyticase）结合Biospin真菌基因组DNA提取试剂盒提取基因组DNA,A260／A280检测纯度并计算质量浓度,用真菌通用引物ITS1／ITS4扩增真菌核糖体基因（rDNA）内转录间区ITS基因,经PCR扩增检验所提取的DNA质量。结果 成功提取所有23株真菌基因组DNA,其纯度及质量浓度能满足PCR反应的要求。结论 用溶细胞酶结合Biospin真菌基因组DNA提取试剂盒从酵母菌、无绿藻及丝状真菌提取的DNA可用于PCR反应。     

Weed control and cover crop management affect mycorrhizal colonization of grapevine roots and arbuscular mycorrhizal fungal spore populations in a California vineyard

Arbuscular mycorrhizal (AM) fungi naturally colonize grapevines in California vineyards. Weed control and cover cropping may affect AM fungi directly, through destruction of extraradical hyphae by soil disruption, or indirectly, through effects on populations of mycorrhizal weeds and cover crops. We examined the effects of weed control (cultivation, post-emergence herbicides, pre-emergence herbicides) and cover crops (Secale cereale cv. Merced rye, × Triticosecale cv.Trios 102) on AM fungi in a Central Coast vineyard. Seasonal changes in grapevine mycorrhizal colonization differed among weed control treatments, but did not correspond with seasonal changes in total weed frequency. Differences in grapevine colonization among weed control treatments may be due to differences in mycorrhizal status and/or AM fungal species composition among dominant weed species. Cover crops had no effect on grapevine mycorrhizal colonization, despite higher spring spore populations in cover cropped middles compared to bare middles. Cover crops were mycorrhizal and shared four AM fungal species (Glomus aggregatum, G. etunicatum, G. mosseae, G. scintillans) in common with grapevines. Lack of contact between grapevine roots and cover crop roots may have prevented grapevines from accessing higher spore populations in the middles.     

Detection of arbuscular mycorrhizal fungal spores in the air across different biomes and ecoregions

Aerial dispersal of fungal spores is common, but the role of wind and air movement in dispersal of spores of arbuscular mycorrhizal (AM) fungi is largely unknown. Several studies have examined the possibility of AM fungal spores being moved by wind vectors without observing spores taken from the air environment. For the first time this study observed the presence of AM fungal spores in the air. The frequency of AM fungal spores in the air was determined in six North American biomes composed of 18 ecoregions. Multiple samples were taken from both the air and the soil at each location. AM fungal spores were found in high abundance in the soil (hundreds of spores per gram of soil), however, they were rarely found in the air (most samples contained no AM fungal spores). Furthermore, only the Glomus morphotype was found in the air, whereas spores in the soil were taxomomically more diverse (Glomus, Acaulospora, Gigaspora, Scutellospora morphotypes were observed). The proportion of Glomus spores in the air relative to Glomus spores in the soil was highest in more arid systems, indicating that AM fungi may be more likely to be dispersed in the air in such systems. Nonetheless, the results indicate that the air is not likely a dominant mode of dispersal for AM fungi.     

Effects of two sterol biosynthesis inhibitor fungicides (fenpropimorph and fenhexamid) on the development of an arbuscular mycorrhizal fungus

The effects of different concentrations (0.2, 2, 20, 200 mg l⁻¹) of two sterol biosynthesis inhibitor (SBI) fungicides, i.e. fenpropimorph and fenhexamid, were evaluated on the spore germination, germ tube elongation, sporulation, and root colonization of Glomus intraradices grown monoxenically in association with transformed carrot roots. The percentage of germinated spores incubated on the SBI fungicides and the length of the germ tubes decreased with increasing concentrations of both fungicides. However, for spore germination this impact was fungistatic rather than fungicidal. Extraradical mycelium architecture and spore production in contact with the SBI fungicides were also strongly impacted at high concentration (20 mg l⁻¹). Conversely, the colonization of roots developing in the fungicide-free compartment, but interconnected with the extraradical mycelium developing on the SBI fungicides, appeared unaffected. Our results demonstrated that the monoxenic culture system could be used as a standardized, reproducible technique to compare the impacts of different molecules on arbuscular mycorrhizal fungi, and for the initial screening of new candidate molecules before registration.