Modifikasi Metode Preparasi Pewarnaan Akar untuk Deteksi dan Visualisasi Pembentukan Koloni Fungi Mikoriza Arbuskula (FMA)

The root staining method was used to detect the presence of Arbuscular Mycorrhizal Fungi (AMF) and to calculate the colonization of the AMF in the roots. Optimal root staining methods not only help in the study of plant-associated mycorrhizae, but also save time and money. Root staining aims to clarify and sharpen the picture of AMF associated with plants on the roots, making it easier to observe with a light microscope. This study aims to obtain an effective (fast, safe, and economical) and flexible method of preparation of root staining so that the visualization of AMF on roots becomes clear and contrasting. The method used in this research is descriptive qualitative which includes the cleanliness of the roots from cell contents, root texture, and color contrast, which consists of three treatments, namely P1 (control) using 10% KOH with 90°C heating and followed by the modified Philips & Hayman staining procedure (1970), P2 using 5% KOH by heating at 90°C followed by the modified Philips & Hayman (1970) procedure using 1% HCl, and P3 using 5% KOH heating at 90°C then stained followed the staining method of Philips & Hayman (1970) which was modified by the use of commercial vinegar as a substitute for HCl. All treatments used Trypan Blue dye. The results showed that the P2 and P3 treatments showed results that were not different from P1, the state of the roots is clean enough thereby detection and visualization of AMF could still be observed properly. The P3 treatment method with heating for 9 minutes can be an alternative method that is effective (fast, safe, economical) and flexible. It faster than common method because it takes 9 minutes for cleaning root cells, relatively safe with the use of commercial vinegar solution to replace HCl solution, and economical because it can reduce the need for KOH material up to 50%.Pewarnaan akar bertujuan untuk memperjelas dan mempertajam gambaran FMA yang berasosiasi dengan tumbuhan pada bagian akar sehingga mempermudah dalam pengamatan dengan mikroskop cahaya. Penelitian dilakukan untuk mendapatkan metode preparasi pewarnaan akar yang efektif (cepat, aman, dan ekonomis) dan fleksibel sehingga visualisasi FMA pada akar menjadi jelas dan kontras. Metode yang digunakan pada penelitian ini adalah deskriptif kualitatif yang mencakup kebersihan akar dari isi sel, tekstur akar, dan kekontrasan warna, yang terdiri dari tiga perlakuan yaitu P1 (kontrol) menggunakan KOH 10% dengan pemanasan 90°C dan diikuti dengan prosedur pewarnaan Philips & Hayman (1970) yang dimodifikasi, P2 menggunakan KOH 5% dengan pemanasan pada suhu 90°C dilanjutkan dengan prosedur pewarnaan yang dimodifikasi dengan memakai HCl 1%, dan P3 menggunakan KOH 5% pemanasan pada suhu 90°C kemudian diwarnai mengikuti metode pewarnaan yang dimodifikasi dengan penggunaan cuka komersial (untuk konsumsi) sebagai pengganti HCl. Semua perlakuan menggunakan pewarna Biru Trypan. Hasil penelitian menunjukkan bahwa perlakuan P2 dan P3 menunjukkan hasil yang tidak berbeda dengan P1 yakni keadaan akar yang cukup bersih sehingga deteksi dan visualisasi FMA masih dapat diamati dengan baik. Metode perlakuan P3 dengan pemanasan selama 9 menit dapat menjadi metode alternatif yang efektif (cepat, aman, ekonomis) dan fleksibel; cepat karena waktu yang diperlukan 9 menit untuk pembersihan sel-sel akar, relatif aman dengan penggunaan larutan cuka komersial (vinegar) untuk menggantikan larutan HCl, ekonomis karena dapat menurunkan kebutuhan bahan KOH hingga 50%

A comparison of methodologies for the staining and quantification of intracellular components of Arbuscular Mychorrizal (AM) fungi in the root cortex of two varieties of winter wheat

© 2019 The Authors. The definitive peer reviewed, edited version of this article is published in Access Microbiology, https://doi.org/10.1099/acmi.0.000083. This is an open-access article distributed under the terms of the Creative Commons Attribution License.Arbuscular Mychorrizal (AM) fungi are one of the most common fungal organisms to exist in symbiosis with terrestrial plants facilitating the growth and maintenance of arable crops. Wheat has been studied extensively for AM fungal symbiosis using the carcinogen trypan blue as the identifying stain for fungal components, namely arbuscles, vesicles and hyphal structures. The present study uses Sheaffer® blue ink with a lower risk as an alternative to this carcinogenic stain. Justification for this is determined by stained wheat root sections (n = 120), with statistically significant increases in the observed abundance of intracellular root cortical fungal structures stained with Sheaffer® blue ink compared to trypan blue for both Zulu (P = 0.003) and Siskin (P = 0.0003) varieties of winter wheat. This new alternative combines an improved quantification of intracellular fungal components with a lower hazard risk at a lower cost.Peer reviewe

Production of micropropagated melon plantlets adapted to saline environment

An experiment was carried out to evaluate the behaviour on salt stress induced by sodium chloride of two melon clones obtained from micropropagation. Since arbuscular mycorrhizal (AM) fungi seem to increase salt tolerance in some crops, at acclimatisation melon plantlets were mycorrhizal with an AM fungus. The root systems of both mycorrhizal and non mycorrhizal plants were exposed to increasing salt concentrations and then placed in distilled water to study the wilting response and the recovery of plants. Observations were made on several morphological parameters, in particular to verify the modification of root system morphology caused by AM fungus, as demonstrated already by several studies. The analysis of infected roots was carried out following Phillips and Hayman's protocol (1970). Afterwards, the specimen was read using an optical microscope

A new process to promote the use of controlled mycorrhization practice in forest nurseries

The aims of this study were to test a new mycorrhizal inoculation process using a “catalyser” of the mycorrhizal establishment (termite mounds of Macrotermes subhyalinus) to minimize the requested volume of fungal inoculum added to the cultural substrate. The effects of the termite mound were explored on mycorrhiza formation between an Australian Acacia, Acacia holosericea and an ectomycorrhizal fungus or an arbuscular mycorrhizal fungus using a two-step cultural system. The first step of this cultural practice was the inoculation of A. holosericea seedlings in small soil volumes (5 L plastic containers planted with 100 pre-germinated seeds) whereas the second one allowed the development of these mycorrhized plants in larger soil volumes (1 L pots planted with one seedling). Termite mound amendment significantly enhanced the mycorrhizal formation from both types of fungal isolates. This stimulating effect could probably be attributed to the introduction via the termite mound of a bacterial group (that is, fluorescent pseudomonads) that could act as Mycorrhiza Helper Bacteria (MHB). Since it is possible to reduce the requested fungal inoculum in controlled mycorrhization practice using M. subhyalinus mound powders, this biotechnological process could be useful in re-afforestation of tropical regions by lowering the requested fungal inoculum quantities and reducing the financial costs of controlled mycorrhization in forest nurseries

Pembersihan Isi Sel Akar dan Jenis Warna Tinta untuk Deteksi Cendawan Mikoriza Arbuskula: Clearing of Root Cell Content and Types of Ink Stain for Arbuscular Mycorrhizal Fungal Detection

Arbuscular mycorrhizal (AM) fungi form mutualistic symbiosis with root of host plant. Staining technique to detect AM fungi usually used hazardous chemical. The ink stain and vinegar were used as an alternative technique to replace trypan blue and lactic acid in root staining method. This study aimed to determine time for clearing root cell contents and ink stain type to visualize the best AM fungal structures within the root observed under light microscope. Pueraria phaseoloides var. javanica roots colonized by AM fungi were cut into 1 cm long, cleared in KOH solution and stained.  Four clearing time were done vis 5, 10, 15 and 20 minutes, and four stains were used namely Shaeffer black ink, Parker Quink blue ink, blue stamp ink, and trypan blue. Twenty stained roots were taken randomly from each tratment, and observed. Root clearing process for 20 minutes showed the best result. Only Shaeffer black ink and trypan blue produced clear structure of external hyphae, internal hyphae, vesicles and arbuscules. Arbuscular structure stained only by Shaeffer black ink and trypan blue. This indicated that Shaeffer black ink could be used as an alternative stain to detect AM fungi within the root of host plantArbuscular mycorrhizal (AM) fungi form mutualistic symbiosis with root of host plant. Staining technique to detect AM fungi usually used hazardous chemical. The ink stain and vinegar were used as an alternative technique to replace trypan blue and lactic acid in root staining method. This study aimed to determine time for clearing root cell contents and ink stain type to visualize the best AM fungal structures within the root observed under light microscope. Pueraria phaseoloides var. javanica roots colonized by AM fungi were cut into 1 cm long, cleared in KOH solution and stained.  Four clearing time were done vis 5, 10, 15 and 20 minutes, and four stains were used namely Shaeffer black ink, Parker Quink blue ink, blue stamp ink, and trypan blue. Twenty stained roots were taken randomly from each tratment, and observed. Root clearing process for 20 minutes showed the best result. Only Shaeffer black ink and trypan blue produced clear structure of external hyphae, internal hyphae, vesicles and arbuscules. Arbuscular structure stained only by Shaeffer black ink and trypan blue. This indicated that Shaeffer black ink could be used as an alternative stain to detect AM fungi within the root of host plan

Staining and microscopy of mycorrhizal fungal colonization in preserved ericoid plant roots

BACKGROUND: Visualization of ericoid mycorrhizal colonization using traditional methods relies on either fresh or KOH stored samples. Increasing interest in studying ericoid mycorrhization has highlighted a need for methods which can be used for preserved samples and are simple to implement with commonly available equipment. OBJECTIVE: The aim of this study was to improve on traditional techniques for staining ericoid mycorrhizal fungi and microscopically visualizing ericoid mycorrhizal roots which have been preserved. METHODS: Ericoid mycorrhizal roots were placed in KOH or frozen at -20 degrees C for long-term storage. Traditional Trypan Blue staining methods were modified to reduce damage to fine mycorrhizal hyphae and cortical cells. A high light-intensity dark-field microscopy technique was applied to clearly visualize stained mycorrhizae. The novel application was compared to other commonly used practices. RESULTS: Trypan Blue staining without KOH storage or clearing allowed for successful staining of ericoid mycorrhizal roots stored at -20 degrees C. The application of high light-intensity dark-field microscopy provided high contrast visualization of mycorrhizal structures. CONCLUSIONS: The modified Trypan Blue staining method was effective on frozen root samples, with dark-field microscopy being particularly effective at visualizing dark colored roots. Advantages to this method are lowcost and relatively fast application time. Therefore, this method is a realistic option for large scale analyses with many samples which require long-term preservation.Peer reviewe

Response of Arbuscular mycorrhizal fungi and Rhizobium inoculation on growth and chlorophyll content of Vigna unguiculata (L) Walp Var. Pusa 151

The aim of the present study was to investigate the effect ofRhizobium and Arbuscular mycorrhizal fungi inoculation, both individually and in combination on growth and chlorophyll content of economically important plant Vigna unguiculata L. A significant (p < 0.05) increase over control in root length (45.6  cm), shoot height (12.2 cm), dry weight of root (0.4 g) and shoot (1.8 g), total number of nodules (39.6 nos.), dry weight of nodules (0.5 g), percentage of mycorrhizal infection (96.6 %), chlorophylls a (0.83 mg/g fr.wt.), b (1.19 mg/g fr.wt) and total chlorophyll (2.24 mg/g fr.wt) was recorded in dual inoculated (AM fungi and Rhizobium) plants than plants with individual inoculation. Thus it is  clear that the dual inoculation with AM Rhizobium biofertilizer is more effective than the individual treatment. @JASEMJ. Appl. Sci. Environ. Manage. December, 2010, Vol. 14 (4) 113 - 11

Ultrastructural changes of tomatoes (Lycopersicon esculentum) root colonized by Glomus mosseae and Ralstonia solanacearum

The colonization of plant root cell by mycorrhizal fungi is one of the mechanisms involved for the understanding of plant bio-protection against soil-borne pathogens. The aim of current study was to investigate and describe tomato (Lycopersicon esculentum) root ultra-structural modifications caused by Glomus mosseae and the bacterial wilt Ralstonia solanacearum. In scanning electron microscopy (SEM) observations, the root cells presented several arbuscules and mature spores of G. mosseae. In transmission electron microscopy (TEM) observations, many entry points on the cell wall were detected in addition to nucleus, cell organs and many mitochondria. The results evidenced that the presence of G. mosseae can change the root architecture dramatically. R. solanacearum was inhibited by the endophytic fungi. G. mosseae structure can help the plant to prevent the pathogen bacterial invasion totally due to root architecture system changes.Key words: Mycorrhizal fungi, bacterial wilt, tomato, root cell, scanning electron microscopy, transmission electron microscopy

Influence of the insecticide dimethoate on arbuscular mycorrhizal colonization and growth in soybean plants

Application to the soil of the insecticide dimethoate had no effect on the growth of soybean colonized by the arbuscular mycorrhizal (AM) fungus Glomus mosseae and by the indigenous AM fungus. The application of the recommended concentration of dimethoate decreased the percentage of colonization of soybean by the indigenous AM population, but no significant effect was observed on the colonization of soybean inoculated with G. mosseae. The insecticide did not affect the germination of G. mosseae spores; however, 0.5 mg/l of dimethoate increased the germination of Gigaspora roseae and 5 mg/l of dimethoate decreased the germination of Scutellospora castaneae spores