Intercellular Hyphae Research Articles

Double-stranded RNA prevents and cures infection by rust fungi

Fungal pathogens that impact perennial plants or natural ecosystems require management strategies beyond fungicides and breeding for resistance. Rust fungi, some of the most economically and environmentally important plant pathogens, have shown amenability to double-stranded RNA (dsRNA) mediated control. To date, dsRNA treatments have been applied prior to infection or together with the inoculum. Here we show that a dsRNA spray can effectively prevent and cure infection by Austropuccinia psidii (cause of myrtle rust) at different stages of the disease cycle. Significant reductions in disease coverage were observed in plants treated with dsRNA targeting essential fungal genes 48 h pre-infection through to 14 days post-infection. For curative treatments, improvements in plant health and photosynthetic capacity were seen 2–6 weeks post-infection. Two-photon microscopy suggests inhibitory activity of dsRNA on intercellular hyphae or haustoria. Our results show that dsRNA acts both preventively and curatively against myrtle rust disease, with treated plants recovering from severe infection. These findings have immediate potential in the management of the more than 10-year epidemic of myrtle rust in Australia.

Synergism study of Bacopa monnieri and Piriformospora indica and its impact on Biomass and metabolite

BackgroundA symbiotic connection between Piriformospora indica and Bacopa monnieri (L.) Wettest, obtained through co-cultivation synergism, was found to improve growth, biomass production, and bacoside content in the plants. Brahmi (B. monnieri L.), a well-known Indian plant prized for its memory-boosting properties, has a lengthy history and a premium price tag. Because of its remarkable ability to colonize a wide variety of plant species, the axenically cultivable mycorrhiza-like endophytic fungus P. indica has gained a lot of interest recently.MethodsIn the current study, fungal spores from recently revived cultures were added to jam bottles next to rooted Brahmi plants for in vitro co-cultivation. The control plants were left without fungal discs. Pre-rooted micro-propagated Brahmi plants were treated with agar discs containing actively growing hyphae. For a period of 3 months, both trials were conducted with a fully randomized setup. Microscopy of the treated and control plant roots verified co-cultivation.ResultsMicroscopic examination of the roots of co-cultivated plants reveals a high degree of colonization with host plants. These endophytic fungal structures include intracellular chlamydospores, and arbuscules, an intercellular and intracellular hyphae network, and a mycelial network on the root surface. In both in vitro and in vivo co-cultivation studies, the plant extended the host plant’s lifespan in 3 months by displaying continuous regeneration; in contrast, the control plant displayed signs of senescence. With biomass exceeding the control by 1.18 times in vivo and 1.28 times in vitro. In vitro, co-cultivation circumstances also led to an increase in the rate of utilization of nutritional medium. In comparison to the control, the amount of bacoside increases to 100% in vivo after a month of co-cultivation and 33% in vitro after 3 months.ConclusionsIn the present investigation, in vivo co-cultivation showed a favorable interaction effect on biomass production as well as bacoside content, which can satisfy the raw material demands of Brahmi plants in pharmaceutical industries.

Austropuccinia psidii infection changes stomatal dynamics and leaf gas exchange of guava leaves: A physio-histopathological assessment

This study aimed to characterise the life cycle of Austropuccinia psidii, the causal agent of myrtle rust, in guava cv. Paluma and to determine how this pathogen affects gas exchange in diseased leaves. The pathogen formed appressoria on ordinary epidermal cells and intercellular hyphae and intracellular branched lobe-shaped haustoria in parenchyma cells. In the stem, hyphae grew in the cortical region and crossed between tracheary cells, reaching the medullary region. In the leaf, hyphae colonised the intercellular spaces of the main vein and mesophyll and the substomatal chamber. Colonisation of the substomatal chamber by A. psidii changed the stomatal dynamics and leaf gas exchange. The stomatal response to light was slower and had a smaller amplitude in diseased guava leaves. Consequently, impaired stomatal movement induced by A. psidii is an additional factor changing the performance of plants under abiotic stress.

Exploring structural and molecular diversity of Ericaceae hair root mycobionts: a comparison between Northern Bohemia and Argentine Patagonia.

Core Ericaceae produce delicate hair roots with inflated rhizodermal cells that host plethora of fungal symbionts. These poorly known mycobionts include various endophytes, parasites, saprobes, and the ericoid mycorrhizal (ErM) fungi (ErMF) that form the ErM symbiosis crucial for the fitness of their hosts. Using microscopy and high-throughput sequencing, we investigated their structural and molecular diversity in 14 different host × site combinations in Northern Bohemia (Central Europe) and Argentine Patagonia (South America). While we found typical ericoid mycorrhiza in all combinations, we did not detect ectomycorrhiza and arbuscular mycorrhiza. Superficial mantles of various thickness formed by non-clamped hyphae were observed in all combinations except Calluna vulgaris from N. Bohemia. Some samples contained frequent intercellular hyphae while others possessed previously unreported intracellular haustoria-like structures linked with intracellular hyphal coils. The 711 detected fungal OTU were dominated by Ascomycota (563) and Basidiomycota (119), followed by four other phyla. Ascomycetes comprised Helotiales (255), Pleosporales (53), Chaetothyriales (42), and other 19 orders, while basidiomycetes Sebacinales (42), Agaricales (28), Auriculariales (7), and other 14 orders. While many dominant OTU from both hemispheres lacked close relatives in reference databases, many were very similar to identical to unnamed sequences from around the world. On the other hand, several significant ericaceous mycobionts were absent in our dataset, incl. Cairneyella, Gamarada, Kurtia, Lachnum, and Leohumicola. Most of the detected OTU could not be reliably linked to a particular trophic mode, and only two could be reliably assigned to the archetypal ErMF Hyaloscypha hepaticicola. Probable ErMF comprised Hyaloscypha variabilis and Oidiodendron maius, both detected only in N. Bohemia. Possible ErMF comprised sebacinoid fungi and several unnamed members of Hyaloscypha s. str. While H. hepaticicola was dominant only in C. vulgaris, this model ErM host lacked O. maius and sebacinoid mycobionts. Hyaloscypha hepaticicola was absent in two and very rare in six combinations from Patagonia. Nine OTU represented dark septate endophytes from the Phialocephala fortinii s. lat.-Acephala applanata species complex, including the most abundant OTU (the only detected in all combinations). Statistical analyses revealed marked differences between N. Bohemia and Patagonia, but also within Patagonia, due to the unique community detected in a Valdivian temperate rainforest. Our results show that theericaceous hair roots may host diverse mycobionts with mostly unknown functions and indicate that many novel ErMF lineages await discovery. Transhemispheric differences (thousands of km) in their communities may be evenly matched by local differences (scales of km, m, and less).

Plant lysin motif extracellular proteins are required for arbuscular mycorrhizal symbiosis

Arbuscular mycorrhizal fungi (AMF) can form a mutually beneficial symbiotic relationship with most land plants. They are known to secrete lysin motif (LysM) effectors into host root cells for successful colonization. Intriguingly, plants secrete similar types of LysM proteins; however, their role in plant-microbe interactions is unknown. Here, we show that Medicago truncatula deploys LysM extracellular (LysMe) proteins to facilitate symbiosis with AMF. Promoter analyses demonstrated that three M. truncatula LysMe genes MtLysMe1/2/3, are expressed in arbuscule-containing cells and those adjacent to intercellular hyphae. Localization studies showed that these proteins are targeted to the periarbuscular space between the periarbuscular membrane and the fungal cell wall of the branched arbuscule. M. truncatula mutants in which MtLysMe2 was knocked out via CRISPR/Cas9-targeted mutagenesis exhibited a significant reduction in AMF colonization and arbuscule formation, whereas genetically complemented transgenic plants restored wild-type level AMF colonization. In addition, knocking out the ortholog of MtLysMe2 in tomato resulted in a similar defect in AMF colonization. In vitro binding affinity precipitation assays suggested binding of MtLysMe1/2/3 with chitin and chitosan, while microscale thermophoresis (MST) assays revealed weak binding of these proteins with chitooligosaccharides. Moreover, application of purified MtLysMe proteins to root segments could suppress chitooctaose (CO8)-induced reactive oxygen species production and expression of reporter genes of the immune response without impairing chitotetraose (CO4)-triggered symbiotic responses. Taken together, our results reveal that plants, like their fungal partners, also secrete LysM proteins to facilitate symbiosis establishment.

Phenolic acids and defense-related enzymes in host-pathogen interaction in Senecio aegyptius and the rust fungus, Puccinia lagenophorae

The objective of this study was to investigate the effect of the rust fungus Puccinia lagenophorae on total phenols, phenolic acids, and defense-related enzymes in the leaf tissues of the host, Senecio aegyptius. The infection increased the total phenol and defense-related enzymes in the host leaf tissues when compared to healthy tissues. The analysis using HPLC revealed the existence of 10 phenolic acids in healthy and infected host leaves. The infection increased the amounts of caffeic, chlorogenic, ferulic, p-coumaric, and salicylic acids. The examination by TEM revealed the presence of peroxidase activity on the host microbodies, Golgi bodies, endoplasmic reticulum, and the thickening of xylem vessels. After the infection, the activity was localized at the penetration sites of fungal haustoria and in amorphous materials found between the intercellular hyphae and the host cell wall. The heaviest peroxidase activity was detected in heavily infected vascular tissue, due to the monokaryotic infection.

Effector-mediated partial and nonhost disease resistance in wheat.

Effector-mediated partial and nonhost disease resistance in wheat.

Double-Staining Method to Detect Pectin in Plant-Fungus Interaction.

Plant cells use different structural mechanisms, either constitutive or inducible, to defend themselves from fungal infection. Encapsulation is an efficient inducible mechanism to isolate the fungal haustoria from the plant cell protoplast. Conversely, pectin, one of the polymeric components of the cell wall, is a target of several pectolytic enzymes in necrotrophic interactions. Here, a protocol to detect pectin and fungal hyphae through optical microscopy is presented. The pectin-rich encapsulation in the cells of coffee leaves infected by the rust fungus Hemileia vastatrix and the mesophyll cell wall modification induced by Cercospora coffeicola are investigated. Lesioned leaf samples were fixed with the Karnovsky solution, dehydrated, and embedded in glycol methacrylate for 2-4 days. All steps were followed by vacuum-pumping to remove air in the intercellular spaces and improve the embedding process. The embedded blocks were sectioned into 5-7 µm thick sections, which were deposited on a glass slide covered with water and subsequently heated at 40 °C for 30 min. Next, the slides were double-stained with 5% cotton blue in lactophenol to detect the fungus and 0.05% ruthenium red in water to detect pectin (acidic groups of polyuronic acids of pectin). Fungal haustoria of Hemileia vastatrix were found to be encapsulated by pectin. In coffee cercosporiosis, mesophyll cells exhibited dissolution of cell walls, and intercellular hyphae and conidiophores were observed. The method presented here is effective to detect a pectin-associated response in the plant-fungi interaction.

Karakteristik Mikoriza Arbuskula Tanaman Serai Wangi (Cymbopogon nardus L.) di Lapangan Ternaungi dan Tidak Ternaungi

Arbuscular mycorrhiza (AM) characteristics of citronella grass in the field have not been reported. This research aimed to study the AM characteristics of citronella grass grown in unshaded and shaded fields. The roots of citronella grass were collected from citronella grass plantations in Cianjur, West Java. The root samples were analyzed for AM structures, namely entry points, intercellular hyphae, arbuscules, and vesicles. The results showed that the citronella grass form AM colonization. The quality of root colonization differed between the two cultivation systems. The unshaded citronella grass had higher root colonization compared to shaded citronella grass. In the unshaded citronella grass, the number of arbuscules was 7 per cm of root length, whereas in the shaded citronella grass was 4 per cm of root length. The types of arbuscules observed were arum and intermediate. There were no differences in the number of entry points in the two cultivated systems, which was 3,5 entry points per cm of root length. The numbers of vesicles and internal hyphae in unshaded citronella grass were lower than that of in the shaded citronella grass. In the unshaded citronella grass, the number of vesicles and intracellular hyphae were 1,5 and 8,5 per cm root length, whereas in the shaded citronella grass were 3,5 and 11 per cm root length, respectively. Shading plants grown in the field were bamboo, banana, coffee, tea, and sugar palm. All the shading plants formed AM symbiosis with a colonization value of 7 to 30%. This research indicates that arbuscular mycorrhiza is an important component in the citronella grass cultivation in unshaded and shaded fields.
 
 Keywords: Arbuscule, entry point, intercellular hyphae, root colonization, vesicle

Kaolin Particle Film Protects Grapevine cv. Cabernet Sauvignon Against Downy Mildew by Forming Particle Film at the Leaf Surface, Directly Acting on Sporangia and Inducing the Defense of the Plant.

Downy mildew is a major threat to viticulture, leading to severe yield loss. The use of traditional copper-based fungicides is effective, but has adverse effects on the environment and human health, making it urgent to develop an environmentally friendly disease management program. Multi-functional kaolin particle film (KPF) is promising as an effective and safer treatment strategy, since this material lacks chemically active ingredients. In this study, ability of Kaolin particle film (KPF) pretreatment to protect grapevine leaves from Plasmopara viticola was tested and the mode of action of KPF was analyzed. KPF application reduced the disease severity and the development of intercellular hyphae. Additionally, there was reduced accumulation of H2O2 and malondialdehyde (MDA) with pretreatment. The observation of ultrastructure on the leaf surface showed KPF deposition and stomatal obstruction, indicating that KPF protected plants against disease by preventing the adhesion of pathogens to the leaf surface and blocking invasion through the stomata. KPF pretreatment also activated host defense responses, as evidenced by increased activities of anti-oxidative enzymes [superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT)] and defense-related enzymes [phenylalanine ammonia-lyase (PAL), chitinases, and β-1,3-glucanases], increased phytohormone signals [abscisic acid (ABA), salicylic acid (SA), and jasmonic acid (JA)] and the up-regulation of defense genes related to plant defense. Overall, these results demonstrate that KPF treatment counters grapevine downy mildew by protecting leaves and enhancing plant defense responses.

Localized expression of the Dwarf14-like2a gene in rice roots on infection of arbuscular mycorrhizal fungus and hydrolysis of rac-GR24 by the encoded protein

ABSTRACT Strigolactones (SLs) are plant hormones that control diverse aspects of the shoot and root growth and are exuded into the soil as recruitment signals for arbuscular mycorrhizal (AM) fungi. SL signaling in plants is transduced via the α/β-hydrolase receptor Dwarf14 (D14). The D14 family consists of D14, Dwarf14-like (D14L), and Dwarf14-like 2 (D14L2) clades in rice. The D14L receptor is known to condition pre-symbiotic perception of AM fungi. In this study, it was found that the Dwarf14-like2a (D14L2a) gene expression was significantly induced by AM fungal colonization. The transcript of D14L2a appeared not only in mature arbuscule-containing cells but also in epidermal/cortical cells at an early colonization stage and near the elongating intercellular hyphae. D14L2a transcript was detected normally in mycorrhizal roots of str1-2 mutant that form stunted arbuscules, suggesting that the gene expression is independent of arbuscule development. Moreover, the recombinant D14L2a protein exhibited hydrolase activity of synthetic SL, rac-GR24. Based on these results, we discussed the role of D14L2 in the establishment of AM symbiosis.

Genome-Wide Analysis of Coding and Non-coding RNA Reveals a Conserved miR164-NAC-mRNA Regulatory Pathway for Disease Defense in Populus.

MicroRNAs (miRNAs) contribute to plant defense responses by increasing the overall genetic diversity; however, their origins and functional importance in plant defense remain unclear. Here, we employed Illumina sequencing technology to assess how miRNA and messenger RNA (mRNA) populations vary in the Chinese white poplar (Populus tomentosa) during a leaf black spot fungus (Marssonina brunnea) infection. We sampled RNAs from infective leaves at conidia germinated stage [12 h post-inoculation (hpi)], infective vesicles stage (24 hpi), and intercellular infective hyphae stage (48 hpi), three essential stages associated with plant colonization and biotrophic growth in M. brunnea fungi. In total, 8,938 conserved miRNA-target gene pairs and 3,901 Populus-specific miRNA-target gene pairs were detected. The result showed that Populus-specific miRNAs (66%) were more involved in the regulation of the disease resistance genes. By contrast, conserved miRNAs (>80%) target more whole-genome duplication (WGD)-derived transcription factors (TFs). Among the 1,023 WGD-derived TF pairs, 44.9% TF pairs had only one paralog being targeted by a miRNA that could be due to either gain or loss of a miRNA binding site after the WGD. A conserved hierarchical regulatory network combining promoter analyses and hierarchical clustering approach uncovered a miR164–NAM, ATAF, and CUC (NAC) transcription factor–mRNA regulatory module that has potential in Marssonina defense responses. Furthermore, analyses of the locations of miRNA precursor sequences reveal that pseudogenes and transposon contributed a certain proportion (∼30%) of the miRNA origin. Together, these observations provide evolutionary insights into the origin and potential roles of miRNAs in plant defense and functional innovation.

Ceratocystis fimbriata Employs a Unique Infection Strategy Targeting Peltate Glandular Trichomes of Sweetpotato (Ipomoea batatas) Plants.

The infection processes of Ceratocystis fimbriata BMPZ13 (BMPZ13) was elucidated on vegetative tissues of sweetpotato plants employing light and scanning electron microscopy. Vegetative tissues infected with C. fimbriata BMPZ13 by either wounding or nonwounding inoculation methods developed typical disease symptoms, establishing black rot in stems and necrosis on buds, young leaves, and stems of sprouts, in addition to wilt on leaves and shoot cuttings, typical of vascular associated diseases. The runner hyphae of C. fimbriata BMPZ13 formed from germinated conidia were able to directly penetrate the epidermal cuticle for initial infection and invade sweetpotato peltate glandular trichomes, specialized secretory structures to store and secrete metabolites. A two-step biotrophic phase was observed with nonwounding inoculation on leaves and stems, featuring both intercellular and intracellular invasive hyphae, with the latter found within living cells of the leaf epidermis. Subsequent to the biotrophic phase was a necrotrophic phase displaying cell death in infected leaves and veins. Additionally, this cell death was an iron-associated ferroptosis, supporting the notion that iron is involved in the necrotrophic phase of C. fimbriata BMPZ13 infection. Significantly, we establish that C. fimbriata employs a unique infection strategy: the targeting of peltate glandular trichomes. Collectively, our findings show that C. fimbriata is a plant fungal pathogen with a hemibiotrophic infection style in sweetpotato vegetative tissues.

First Cryo-Scanning Electron Microscopy Images and X-Ray Microanalyses of Mucoromycotinian Fine Root Endophytes in Vascular Plants.

AimsArbuscule-producing fine root endophytes (FRE) (previously incorrectly Glomus tenue) were recently placed within subphylum Mucoromycotina; the first report of arbuscules outside subphylum Glomeromycotina. Here, we aimed to estimate nutrient concentrations in plant and fungal structures of FRE and to test the utility of cryo-scanning electron microscopy (cryoSEM) for studying these fungi.MethodsWe used replicated cryoSEM and X-ray microanalysis of heavily colonized roots of Trifolium subterraneum.ResultsIntercellular hyphae and hyphae in developed arbuscules were consistently very thin; 1.35 ± 0.03 μm and 0.99 ± 0.03 μm in diameter, respectively (mean ± SE). Several intercellular hyphae were often adjacent to each other forming “hyphal ropes.” Developed arbuscules showed higher phosphorus concentrations than senesced arbuscules and non-colonized structures. Senesced arbuscules showed greatly elevated concentrations of calcium and magnesium.ConclusionWhile uniformly thin hyphae and hyphal ropes are distinct features of FRE, the morphology of fully developed arbuscules, elevated phosphorus in fungal structures, and accumulation of calcium with loss of structural integrity in senesced arbuscules are similar to glomeromycotinian fungi. Thus, we provide evidence that FRE may respond to similar host-plant signals or that the host plant may employ a similar mechanism of association with FRE and AMF.

Arbuscular Mycorrhizal and Dark Septate Endophyte Fungal Association in Cassava (Manihot esculenta Crantz) Varieties, Southern India

The study examined the root characteristics, type of arbuscular mycorrhizal (AM) and dark septate endophyte (DSE) fungal associations and their extent of colonization in 13 widely cultivated cassava (Manihot esculenta Crantz) varieties in southern India. Root and associated soil samples of each cassava variety were collected from Salem and Kolli Hills of the Eastern Ghats in south India. In addition, we also investigated the relationship between the extent of colonization of AM, DSE fungal and AM fungal spore communities and soil characteristics for each cassava variety. Roots of all the cassava varieties were colonized by AM fungi and 12 varieties had co-colonization of DSE fungi. The AM morphology in all the cassava varieties was of an intermediate type with intercellular and intracellular linear hyphae and arbusculate coils. In addition, we found high AM colonization rates (73-89%) but only moderate rates by DSE (22-68%) fungi. We also found 17 AM fungal spore morphotypes and their abundance varied significantly among cassava varieties. In addition, different physicochemical properties of soils may possibly increase AMF diversity, while different varieties of cassava are commonly associated with differing levels of AM and DSE fungi colonization with soil factors potentially influencing the symbiosis.

Structural plasticity in root-fungal symbioses: diverse interactions lead to improved plant fitness.

Root-fungal symbioses such as mycorrhizas and endophytes are key components of terrestrial ecosystems. Diverse in trophy habits (obligate, facultative or hemi-biotrophs) and symbiotic relations (from mutualism to parasitism), these associations also show great variability in their root colonization and nutritional strategies. Specialized interface structures such as arbuscules and Hartig nets are formed by certain associations while others are restricted to non-specialized intercellular or intracellular hyphae in roots. In either case, there are documented examples of active nutrient exchange, reinforcing the fact that specialized structures used to define specific mycorrhizal associations are not essential for reciprocal exchange of nutrients and plant growth promotion. In feremycorrhiza (with Austroboletus occidentalis and eucalypts), the fungal partner markedly enhances plant growth and nutrient acquisition without colonizing roots, emphasizing that a conventional focus on structural form of associations may have resulted in important functional components of rhizospheres being overlooked. In support of this viewpoint, mycobiome studies using the state-of-the-art DNA sequencing technologies have unearthed much more complexity in root-fungal relationships than those discovered using the traditional morphology-based approaches. In this review, we explore the existing literature and most recent findings surrounding structure, functioning, and ecology of root-fungal symbiosis, which highlight the fact that plant fitness can be altered by taxonomically/ecologically diverse fungal symbionts regardless of root colonization and interface specialization. Furthermore, transition from saprotrophy to biotrophy seems to be a common event that occurs in diverse fungal lineages (consisting of root endophytes, soil saprotrophs, wood decayers etc.), and which may be accompanied by development of specialized interface structures and/or mycorrhiza-like effects on plant growth and nutrition.

Histological and biochemical aspects of compatible and incompatible wheat- Puccinia striiformis interactions

The resistant, slow and fast-rusting cultivars were selected from 15 wheat cultivars to study biochemical and histological aspects of compatible and incompatible wheat-Puccinia striiformis interactions. Final rust severity (%) and area under disease progress curve were significantly decreased in resistant cultivars compared with susceptible ones. Biochemical analysis revealed that reactive oxygen species (ROS), i.e. superoxide (O2•-) and hydrogen peroxide (H2O2) significantly accumulated in the susceptible cultivars. While, activities of catalase (CAT), peroxidase (POX) and chlorophyll concentrations were increased in resistant cultivars. Histological examination using scanning and transmission electron microscope indicated that approximately all haustoria and hyphae were inhibited in resistant cultivar (Misr-1), development of haustorial mother cells and haustoria were retarded, and they were abnormal. On the other hand, large amounts of intercellular hyphae presented in the intercellular spaces and many haustoria were produced in fast-rusting cultivar (Sids-12). Nevertheless, a little of sours and spores on upper epidermis and in mesophyll tissue were observed in slow-rusting cultivar (Sakha-93)..

Histopathologic characterization of the process of Marssonina brunnea infection in poplar leaves

Marssonina brunnea (Ellis & Everh.) Magnus, the causative pathogen of Marssonina leaf spot of poplars (MLSP), can lead to complete defoliation and tree death. Although MLSP has been studied for over 30 years, its precise process of infection is currently unclear. In this study, we present the process of M. brunnea infection in detail using several types of microscopy. When the conidia came into contact with the poplar leaves, they developed germ tubes to attach to the leaf surface. During the first 2 days post inoculation (dpi), infection vesicles (IV) and primary hyphae (PH) formed within host cells. The plasma membranes of the host cells penetrated by IV remained unbroken and intact organelles were visible, indicating that the IV did not kill the host cell. At 3 dpi, secondary hyphae (SH) began to appear within and outside the host cells. The ultrastructural evidence indicated that the SH could kill the host cells rapidly, producing black spots on the surfaces of the leaves. These results collectively suggested that M. brunnea is a typical hemibiotrophic fungus. In addition, M. brunnea could develop intercellular infective hyphae (IH) for expansion. Our findings also confirmed that the germ tubes, IV, and SH are crucial structures for the disease interactions and showed that the two formae speciales of M. brunnea share the same infection histopathological features. This provides important insight for further research into M. brunnea.

Evaluation of Growth and Morphological Pattern of Mycorrhization in Cowpea [Vigna unguiculata (L.)] Fertilized With Phosphorus

Microorganisms perform important functions in the soil and, among these organisms, the role of arbuscular mycorrhizal fungi (AMF) in plant growth should be highlighted. AMF colonize the roots of most plant species and their beneficial functions in plant development include increased absorption of nutrients from the soil, especially those of low mobility such as phosphorus (P). Evaluating agricultural practices conducted by farmers, such as phosphate fertilization, and observing how they will influence AMF activity in benefiting plant growth should be prioritized. Thus, an experiment was conducted in greenhouse to evaluate the effect of phosphate fertilization on the growth of cowpea plants colonized by AMF and to know which morphological pattern of colonization prevails in their roots. Five P doses and a control treatment, without fertilization, were added to the soil. Cowpea plants respond to phosphate fertilization up to the dose of 240.50 mg P kg-1 soil, for shoot dry mass and in the dose of 150 mg P kg-1 soil, for plant height. The morphological pattern observed in the roots was the intermediate type, characterized by the presence of intra and intercellular hyphae and vesicles, and there was no influence of phosphate fertilization on morphology. High P contents added to the soil led to a reduction in mycorrhizal colonization in cowpea roots.

Localization of calreticulin and calcium ions in mycorrhizal roots of Medicago truncatula in response to aluminum stress

Aluminum (Al) toxicity limits growth and symbiotic interactions of plants. Calcium plays essential roles in abiotic stresses and legume-Rhizobium symbiosis, but the sites and mechanism of Ca2+ mobilization during mycorrhizae have not been analyzed. In this study, the changes of cytoplasmic Ca2+ and calreticulin (CRT) in Medicago truncatula mycorrhizal (MR) and non-mycorrizal (NM) roots under short Al stress [50 μM AlCl3 pH 4.3 for 3 h] were analyzed.Free Ca2+ ions were detected cytochemically by their reaction with potassium pyroantimonate and anti-CRT antibody was used to locate this protein in Medicago roots by immunocytochemical methods.In MR and NM roots, Al induced accumulation of CRT and free Ca2+. Similar calcium and CRT distribution in the MR were found at the surface of fungal structures (arbuscules and intercellular hyphae), cell wall and in plasmodesmata, and in plant and fungal intracellular compartments. Additionally, degenerated arbuscules were associated with intense Ca2+ and CRT accumulation. In NM roots, Ca2+ and CRT epitopes were observed in the stele, near wall of cortex and endodermis.The present study provides new insight into Ca2+ storage and mobilization in mycorrhizae symbiosis. The colocalization of CRT and Ca2+ suggests that CRT is essential for calcium mobilization for normal mycorrhiza development and response to Al stress.