Biological control of sclerotial blight of tea using arbuscular mycorrhizal fungus and plant growth promoting rhizobacterium.

استفاده از پتانسیل میکروبی خاک از جمله قارچ های میکوریز آربوسکولار (AMF)برای بهبود رشد و تغذیه گیاه بطور گسترده ای مورد توجه قرار گرفته است. این مطالعه به منظور بررسی تأثیر قارچ های میکوریز آربوسکولار بر رشد و درصد مواد مؤثره نعناع فلفلی تحت شرایط گلخانه ای در قالب طرح کاملا تصادفی با 4 تیمار قارچ میکوریز آربوسکولار انجام شد. تیمارهای تلقیحی شامل 1) شرایط بدون تلقیح (C)، 2) گلوموس فسیکولاتوم (Gf)، 3) گلوموس اینترارادیسز (Gi) و 4) گلوموس موسه (Gm) بودند. در پایان دوره رشد برخی پارامترهای رشدی شامل ارتفاع، قطر ساقه، تعداد شاخه جانبی، تعداد برگ، وزن تر و خشک برگ و همچنین درصد مواد مؤثره و درصد کلنیزاسیون ریشه نعناع فلفلی اندازه گیری شدند. نتایج نشان داد تلقیح با قارچ های میکوریز آربوسکولار اثر معنی داری (05/0P≤)، بر روی پارامترهای اندازه گیری داشته است. تلقیح با قارچ های میکوریز آربوسکولار، ارتفاع گیاه، قطر ساقه، تعداد شاخه های جانبی، تعداد برگ، ماده تر و خشک برگ، درصد کلونیزاسیون ریشه و محتوی (25 درصد) و عملکرد اسانس (28 درصد) را افزایش داد. منتول، منتون و 1و 8-سینئول بالاترین فراوانی را در مواد موثره نعناع تشکیل دادند. تأثیر قارچ-های میکوریز آربوسکولار بر وزن خشک ریشه و درصد کلونیزاسیون ریشه معنی دار (05/0P≤) بود. بیشترین و کمترین درصد کلونیزاسیون ریشه (به ترتیب 47 و 0 درصد) در گیاهان تلقیح شده با Gf و تیمار شاهد مشاهده گردید.

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

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.

Arbuscular mycorrhizal (AM) fungi are soil borne fungi forming symbiotic relationship with majority of higher plants providing a direct link between soil and plant roots. AM fungi undergo a multi-step colonization process before benefiting the host plant. The strong glue glomalin is a glyco protein produced by an AM fungi. The deposition of glomalin on soil particles leads to buildup and stabilization of aggregates, which leads to better soil structure which in turn leads to increased soil water availability under water stress condition. The mycorrhizal colonization is effective aggregators and therefore management of mycorrhizal fungi can be considered as a biological amendment for soil structure and other soil physical properties. Mycorrhizal plants could take up more metal nutrients via extraradical hyphae, which provide larger surface areas than the roots alone and reduce the distance for diffusion, thereby enhancing the absorption of immobile metal nutrients (especially Phosphorus and Zinc). Mycorrhizal inoculated plants produce larger biomass as a direct consequence of improved photosynthetic activities and translocate 20 to 30% of the assimilated C to the underground where mycorrhizal structures conserve soil carbon. AM symbiosis enhanced the plant growth by increasing plant access to immobile mineral ions mainly Phosphorus and Zinc, improving physical conditions and by binding heavy metals into roots that restricts their translocation into shoot tissues. The mycorrhizal symbiosis assists in biofortification of micronutrients such as Fe and Zn in maize grain irrespective of calcareous and non-calcareous soils. The mechanism involved in improved micro nutrition of maize includes acidification of rhizosphere; siderophore production enhanced physiologically active Fe and production of anti-oxidants besides synergistic interaction between P and micronutrients. Key words: Mycorrhiza, soil aggregation, micronutrients, carbon sequestration, heavy metals, enzymatic activity, glomalin, biofortification.

به منظور مطالعه تأثیر دزهای مختلف علف‌کش ترفلان و دو گونه قارچ‌ میکوریزایی بر اجزاء سبز شدن (سرعت سبز شدن، یکنواختی سبز شدن و زمان تا 10، 50 و 90 درصد سبز شدن)، برخی صفات رشدی و کلونیزاسیون میکوریزا در گیاه شبدر سفید (Trifolium repens L.)، آزمایشی به-صورت فاکتوریل در قالب طرح بلوک های کامل تصادفی در سه تکرار در دانشکده کشاورزی دانشگاه شاهرود در سال 1391 انجام شد. عوامل مورد بررسی در این آزمایش شامل کاربرد میکوریزا در سه سطح M1: عدم تلقیح، M2: Glommus intraradices و :M3 Glommus mosseae و دزهای مختلف علف‌کش ترفلان در چهار سطح T1: شاهد عدم کاربرد علف‌کش، T2: 1000،:T3 1500 و T4 : 2000 میلی‌لیتر علف‌کش ترفلان از نوع تجاری در هکتار انجام شد. نتایج نشان داد با افزایش دز علف‌کش ترفلان، یکنواختی سبز شدن (EU) کاهش و زمان تا رسیدن به 10(D10) و 90(D90) درصد سبز شدن افزایش یافت. در غلظت های مختلف علف‌کش، تلقیح با میکوریزا حداکثر جوانه زنی (Emax) و وزن خشک اندام هوایی را بطور معنی داری نسبت به عدم تلقیح افزایش داد. همچنین، درصد کلونیزاسیون ریشه در دز پایین علف‌کش تحت تاثیر تلقیح میکوریزا نسبت به عدم تلقیح بطور معنی داری افزایش یافت. تلقیح با قارچ G. intraradices طول ریشه را در دزهای 1000 و 1500 میلی لیتر علف‌کش نسبت به عدم تلقیح در همین تیمارها به ترتیب 34 و 57 درصد افزایش داد. بر اساس نتایج این پژوهش، قارچ های میکوریزا از طریق بهبود رشد گیاه سبب کاهش اثرات تنش دزهای پایین علف کش ترفلان بر گیاه شبدر می شوند.

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

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

The application of microbial inoculants (biofertilizers) is a promising technology for future sustainable farming systems in view of rapidly decreasing phosphate stocks and the need to more efficiently use available nitrogen (N). Various microbial taxa are currently used as biofertilizers, based on their capacity to access nutrients from fertilizers and soil stocks, to fix atmospheric nitrogen, to improve water uptake or to act as biocontrol agents. Since the results of biofertilization in the field are inconsistent we conducted a meta-analysis to quantify benefits of biofertilizers in terms of yield increase, nitrogen and phosphorus use efficiency, based on 171 peer reviewed publications that met the eligibility criteria. Major findings are: i) the superiority of biofertilizer performance in dry climates over other climatic regions; ii) yield response due to biofertilizer application was generally small at low soil P levels; efficacy increased along higher soil P levels in the order arbuscular mycorrhizal fungi (AMF), P-solubilizers and N-fixers; iii) success of inoculation with AMF was greater at low organic matter content and at neutral pH. Our comprehensive analysis provides a basis and guidance for proper choice and application of biofertilizers. Rainfed farms on marginal lands will be most affected by scarcity of non-renewable resources such as fertilizers. Mutualistic root organisms like AMF can substantially contribute to a more resilient, sustainably intensified dryland farming system. We are interested to study the possibility to use AMF as “biofertilizers” in an intercropping system in Indian agriculture, planting pigeon pea (Cajanus cajan) seedlings pre-inoculated with AMF into a field sown with finger millet (Eleusine coracana). By destructive sampling over five weeks we estimated a hyphal growth of 4.1mm d-1 by C. etunicatum which is 1mm faster per day than all other estimates. To study the potential of Rhizophagus fasciculatus, Claroideoglomus etunicatum and Rhizophagus intraradices to spread from AMF-inoculated pigeon pea to un-inoculated finger millet seedlings, we established experimental microcosms in the greenhouse, in which the pigeon pea and two finger millet plantlets were kept in separate pots, connected by soil bridges of 5 or 12 cm length inaccessible to roots but accessible to fungal hyphae. We found that depending on the distance different AMF were promoting the growth of finger millet better. We also detected transport of fertilized nitrogen along the hyphae via stable isotope analysis over a distance of up to 12 cm. However these results also depended on the AMF species. We conclude that the row distance between the crops and the choice of AMF species play a crucial role for the application of AMF as biofertilizer and their growth promotion. To understand the effects the biofertilizers Pseudomonas fluorescens and two AMF species on the microbial community in the soil, both the bacterial community and the community of AMF were studied. Samples were collected at harvest from mono- and intercropped pigeon pea and finger millet at two field sites in South India at the University of Agricultural Sciences, GKVK campus, Bangalore and Kolli hills, Tamil Nadu state, India. DNA was extracted from rhizosphere soil surrounding the roots. To detect changes in the bacterial community automated ribosomal intergenic spacer analysis (ARISA) was conducted and treatments were compared using principal component analysis. The strongest effect was found to be exerted by the plant species; biofertilization had no effect on the bacterial community. To detect changes in the AMF community we amplified the whole ITS ribosomal unit and sequenced the barcoded samples with the PacBio platform. Although OTUs from Glomeromycota were found, the sequencing depth remained too little to make firm conclusions about the changes in the AMF community. Our second goal was to trace the applied inoculum at harvest and, although only few sequences were recovered, the inoculum of Rhizophagus fasciculatus could be traced in some treatments.

Management of Stem-rot of Groundnut (Arachis hypogaea L.) Cultivar in Field

Root and rhizosphere soil samples of medicinal plant Huangshan Magnolia (Magnolia cylindrica) from Chinese famous national forest park of Huangshan (Yellow Mountain) were studied to determine the root colonization and the diversity of spore populations of arbuscular mycorrhizal (AM) fungi. The results showed that AM fungal colonization structures including hyphae, hyphal coils and vesicles were present in all root samples. Paris-type AM were identified in the roots according to the morphological structure.Seventeen species of AM fungi were isolated and identified from the rhizosphere soil samples. The species were of the genera Acaulospora (6 species), Glomus (8 species),Gigaspora (1 species) and Scutellospora (2 species). Based on importance value, 3 species from Acaulospora and 3 from Glomus were dominant. The AM fungi spore density ranged from 157 to 448 (average 315) per 100 g soil and the species richness ranged from 4 to 8 (average 6.5) per soil sample. Shannon-Wiener index and Evenness were calculated to evaluate the diversity of the AM fungi community associated with M. cylindrica. Key words: Magnolia cylindrica, arbuscular mycorrhizal fungi, diversity.

Previous studies documented that most desert plants can be colonized by arbuscular mycorrhizal (AM) fungi, however, little is known about how the dynamics of AM fungi are related to ephemerals in desert ecosystems. The dynamics of AM fungi with desert ephemerals were examined to determine the effects of host plant life stages on the development of AM fungi. Mean colonization of ephemeral annual plants was 45% lower than that of ephemeral perennial plants. The colonizations were much higher in the early part of the growing season than in later parts, peaking at flowering times. The phenology of AM fungi in root systems varied among different ephemerals. The density of AM fungal spores increased with the development of ephemeral annual plants, reached its maximum at flowering times, and then plateaued about 20 days after the aboveground senescence. A significant positive correlation was found between AM fungi spore density and biomass of ephemeral annual plants. The life cycles of AM fungi associated with desert ephemerals were very short, being about 60–70 days. Soil temperature and water content had no direct influence on the development of AM fungal spores. We concluded that the development of AM fungi was in response to desert ephemeral phenology and life history strategy.

Plant-driven genome selection of arbuscular mycorrhizal fungi.

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

Molecular ecological analyses of specific interactions between symbionts in the arbuscular mycorrhizal symbiosis

بهبود جذب عناصر غذایی با راهکارهای بیولوژیک، ضمن کمک به تحقق کشاورزی پایدار سبب افزایش و یا ثبات عملکرد گیاهان زراعی می‌گردد. این مطالعه با هدف بررسی اثر تلقیح بذور ژنوتیپ‌های تیپ کابلی نخود با میکوریزای آرباسکولار و شبه‌میکوریزای داخلی بر عملکرد گیاه به‌صورت اسپیلت پلات در قالب طرح بلوک‌های کامل تصادفی در سه تکرار در مزرعه تحقیقاتی دانشگاه فردوسی مشهد به اجرا درآمد. فاکتور اصلی شامل سه سطح میکوریزایی (میکوریزای آرباسکولار Glomus mosseae، شبه‌‌میکوریزای داخلی Piriformosporaindica و عدم مصرف قارچ) و فاکتور فرعی شامل 9 ژنوتیپ نخود (MCC80، MCC358، MCC361، MCC392، MCC427، MCC537، MCC693، MCC696 و MCC950) بود. در این مطالعه صفات عملکرد دانه و میزان عناصر ماکرو و میکرو و پروتئین بافت‌های گیاهی مورد اندازه‌گیری قرار گرفتند. نتایج نشان داد که میکوریزای آرباسکولار به‌طور معنی‌داری باعث افزایش عملکرد دانه (به میزان 1682 کیلوگرم در هکتار) گردید. در بین ژنوتیپ‌های مورد بررسی، بیشترین عملکرد دانه به ژنوتیپ MCC537 اختصاص داشت. میکوریزای آرباسکولار به‌طور معنی‌داری سبب بهبود جذب عناصر نیتروژن، فسفر، پتاسیم، آهن و منگنز گردید. در حالی‌که سطوح میکوریزا تأثیر معنی‌داری بر روی جذب عناصر مس و روی نداشت. ژنوتیپ‌های MCC537، MCC427، MCC80 و MCC392 به‌طور معنی‌داری در جذب عناصر ماکرو موفق‌تر بودند. اما از نظر جذب عناصر میکرو اختلاف معنی‌داری بین ژنوتیپ‌های نخود وجود نداشت.