سعیده رجایی

Forest ecosystems are the main reservoirs of global biodiversity. Microorganisms play a key role in the biochemical cycles of these ecosystems and are used to protect plants and stimulate their growth. Due to the limitations in the application of classic methods in the isolation and identification of endophytic microorganisms, their potentials and functions have yet to be properly understood. In this study, a culture-independent method was used to study the endophytic bacterial communities of some wild legumes. To this end, the Zagros forests of Kermanshah were surveyed and two varieties of Astragalus ovinus and Vicia lutea were sampled. Total DNAs from different parts of the plant samples were extracted and the bacterial 16S rDNA genes were subsequently amplified. Sequencing analysis using the Illumina MiSeq platform showed the most abundant endophytic bacteria belonged to seven orders of Rhizobiales, Xanthomonadales, Sphingomonadales, Pseudomonadales, Chitinophagales, Enterobacteriales, Betaproteobacteriales. The latter were comprised to three classes of Gammaproteobacteria, Alphaproteobacteria and Bacteroidia, and two phyla of Proteobacteria and Bacteroidetes. Our findings showed that members of Proteobacteria were the dominant bacterial endophytes and accounted for more than 99% and 68% of the total population of endophytic bacteria in V. lutea and A. ovinus, respectively. These bacteria, with the highest percentage of taxonomic units (OTUs), have the highest potential to use as biological control agents of crop legumes diseases. This study provides a clear picture of the population, dominance and diversity of bacterial endophytes of wild legumes in the Zagros forests using metagenomic analysis.

Citrus greening (HLB) is one of the most damaging citrus diseases worldwide. As well, the three most important causal agents of this disease in the world include "Candidatus (Ca.) Liberibacter asiaticus ("CLas"), Ca. Liberibacter africanus ("CLaf"), and Ca. Liberibacter americanus ("CLam"). Accordingly, these were reported from Asian-American, African, and American countries, respectively. Of note, "CLas" species is found as the most important causal agent of this disease in all countries in the world, including Iran. It seems that the most effective way to reduce HLB is finding those cultivars that are resistant or tolerant to HLB. Accordingly, in the present study, an attempt was made to evaluate the reaction of a number of common citrus cultivars to this bacterium.

In the present research, ten commercial rootstocks and cultivars of the common citrus in the south of Iran, including Mexican lime, sour orange, mandarin orange, Kinnow mandarin, Orlando tangelo mandarin, local orange, Valencia orange ,sweet lemon, grapefruit, and Lisbon lemons were prepared as seedlings. After checking their health, they were inoculated with the citrus greening agent and then kept in an insect-free greenhouse at night at 25°C and during the day at 30°C.By passing two months from the inoculation, the pathogen was detected by performing specialized tests in inoculated seedlings. Thereafter, three two-year-old seedlings from each one of the above-mentioned rootstocks and cultivars were grafted with two infected buds. Two months after the inoculation, normal and nested PCR tests were performed on the vein tissue extract of the infected seedlings, once every two months, for a 12-month duration. Finally, both sensitivity and relative resistance of cultivars were evaluated based on the average number of days until the detection of the first Ca. L. asiaticus by PCR and also the incubation period of the disease, in each seedling.

The causal agent of this disease ("CLas") was detected at the earliest time(in Valencia oranges and Orlando tangelo mandarin after 180 and 200 days, respectively) after the inoculation with infected graft, compared to other cultivars and rootstocks. Among the studied species, the "CLas" was detected in both Mexican lime and Lisbon lemon with the longest delay (by passing 330 and 300 days from the inoculation, respectively). According to the results, "CLas" was detected in sour orange and sweet lemon 290 and 280 days after the inoculation with infected graft, respectively. Based on the average number of days until the detection of the first Ca. L. asiaticus by PCR and also the incubation period of the disease, in each seedling, Mexican lime and Lisbon lemon were found as the most tolerant species and Valencia oranges and Orlando tangelo mandarin were found as the most susceptible ones to HLB. Moreover, sour orange and sweet lemon seedlings were moderately susceptible ones; and grapefruit, local orange, local, and Kinnow mandarin were moderately tolerant to HLB.

Due to the reason that this bacterium does not grow in conventional medium, in this study, it was not possible to multiply it in artificial medium and then contaminate the plant with it. Therefore, performing the inoculation through grafting the seedlings with HLB infected buds was considered as the most appropriate method for seedlings inoculation with the pathogen. The faster the pathogen can settle and multiply and the symptoms appear in the host, the greater the susceptibility of the host to the disease. Early pathogen’s detection and symptoms’ observation in some hosts such as Valencia oranges and Orlando tangelo mandarin, indicate better bacterial proliferation in both cultivars. Notably, no symptoms of the disease were observed in Mexican lime and Lisbon lemons after 15 months, and one year after the inoculation in sour orange and sweet lemon plants. There is evidence that all known citrus cultivars are affected to this pathogen with different degrees of susceptibility. Based on the results of this study and the results of previous researchers in the world, it seems that researchers should look for the sources of resistance factors to HLB in more species and cultivars. Correspondingly, these sources can be used in the following two ways: used as a rootstock or scion, and used in the efforts of breeders to produce a resistant cultivar.

Common bacterial blight disease (CBB) of bean (Phaseolus vulgaris) caused by Xanthomonas phaseoli (Xp) is considered as one of the most deleterious pathogens for bean production in the world. In this study, 105 samples were collected from asymptomatic wild fabaceous plants, i.e. Astragalus ovinus, Vicia villosa and Vicia lutea, grown in Zagros forests of Iran. The plant samples were cultured on nutrient agar and purified. The isolates were then screened for some important criteria for biological control such as phosphate solubilization, protease activity, IAA and H2S production, and antagonistic effect. Three endophytic bacterial isolates were found as potential biocontrol agents against Xp. Based on key biochemical tests and comparative analysis of the partial 16S rDNA sequences, the isolates were identified as Pseudomonas fluorescens and two Bacillus species. Under greenhouse conditions, all the three strains significantly increased shoot and root lengths in bean plants at the 5% level (P < 0.05) and decreased disease severity above 70%. This is the first report on the presence and capabilities of endophytic bacteria from wild leguminous plants in the Zagros Mountain steppe forests of Iran.

Due to their various roles in the plants’ life, the Arbuscular mycorrhizal fungi (AMF) has been studied from various aspects. In Iran, study on this type of fungi is still at its infancy. Also, the variety of plant species and populations in Iran necessitates a more in-depth analysis compared to other countries in the world. The present study aimed at investigation of the effects of grazing intensity on the diversity and populations of these microorganisms that are symbiotic with the plants and also determining the factors affecting the soil. Three rangelands were considered with three different grazing pressures: no grazing pressure, managed grazing and heavy grazing. Following the sampling of the soil around the rhizosphere of the dominant plant species, the samples were transferred to the laboratory to be analyzed. The results of this study revealed the significant differences in the diversity of AMF species in different sites, and the highest frequency percentage was related to the Septoglomusconstrictum species identified in the heavy grazing region(42.2%).Also, grazing caused changes in the mycorrhizal symbiosis percentage and these factors had a direct correlation, and this could be an evidence of the reduced diversity of AMF, because despite of the existence of spores in the soil, each plant species can only correlate with specific species of AMF. It can be said that appropriate grazing cannot have a negative effect on the population and diversity of AMF, because species diversity indices in a managed grazing region are in a stable state; and, by the proper management of the grazing system and preventing the premature or excessive grazing, its harmful effects can be prevented.

The aim of this study was to examine the symbiosis relationshipbetween some arbuscular mycorrhizal fungi (AMF) and Salsola laricina (Chenopodiaceae), a non-mycotrophic plant speciesand its effect on improving plant growth parameters. Initially, the development of AMF density was monitored through two parameters including evaluation of mycorrhizal colonization of plant roots and density measurement in the soil under field conditions. Then, the spores were counted and the highest four morphotypes were isolated for morphological identification and preparation of inoculum to culture. The seeds of S. laricana were planted inside pots under greenhouse conditions and were inoculated by four isolates of AMF including Archaeospora schenckii, Glomus deserticola, Scutellospora erythropa, and Septoglomus constrictum and one treatment remained non-inoculated as control. High root colonization of the plants was found at six months after inoculation (46%) where the highest morphotypes density belonged to S. constrictum (No./20 g soil-1), suggesting the importance of AMF for plant growth efficiency. The AMF symbiosis generally improved the growth related to the height and weight of shoot and root of S. laricana that were significantly increased. The results led to the conclusion that, identification of interactions between plants, soil properties, and AMF colonization can contribute to improve management of ecosystems.

The lack of a grazing system management program along with other natural and human factors in the country has led to the degradation of rangelands to a large extent. From a new perspective, the lack of grazing management, as an indirect consequence, but with a key role, can have negative effects on useful soil microorganisms, in particular, plant symbiosis with arbuscular mycorrhizal fungi. In this case, due to the sever imbalance between plant and fungus, the reclamation of rangelands with the help of symbiotic soil fungi will be difficult. To investigate the effects of grazing management on AMF symbiosis with S. laricina in three sites of heavy grazing, light grazing, and no grazing, the root and soil were completely randomly sampled around the rhizosphere. The mean of root colonization percentage and soil spores frequency, after assurance of homogeneity of variance and normal mean of data, were compared with the Duncan's analysis of variance at 95% confidence level. Early grazing and the lack of grazing systems management are among the factors that greatly reduce the fungus symbiosis with the roots of plants. Over time, the loss of this important ecosystem chain along with other factors, such as climate change and mismanagement of natural areas, has led to the decreased number of Salsola individuals. This results in changes in species composition and abundance as well as the presence of unpalatable and invasive species, which ultimately leads to the rangeland degradation.

Phytoremediation efficiency -a cost effective and environmentally sound technology to clean up contaminated soil - is reduced considering abiotic stresses. The research objective was to determine the effects of aged petroleum contaminated soil (10% W/W: Total petroleum Hydrocarbons) on two grasses specious (wild oat and barley) during the phytoremediation process. In addition the effects of petroleum degrading rhizobacteria inoculation were evaluated on plant growth promotion and decreasing toxicity of petroleum hydrocarbons. Results demonstrated that petroleum contaminated soil reduced growth, photosynthetic pigments and increased hydrogen peroxide (H2O2), MDA (malondialdehyde), enzymatic antioxidants (catalase and peroxidase) and NCDE and COR2 gene transcripts that indicate oxidative stress happened because of petroleum pollution similar to other common abiotic stress. a/b chlorophyll ratio enhancement and more decrease in shoot dry weight in wild oat showed barley was more tolerate to petroleum pollution. Bacterial inoculation reduced petroleum contamination toxicity and improved plant growth especially in wild oat.

The objectives of this study were isolating rhizosphere-inhabiting indigenous oil-degrading bacteria in wild oat grown in petroleum-polluted areas and in vitro evaluating the efficiency of oil biodegradability by microbial isolates.. Bacteria were isolated from rhizosphere of wild oat grown on contaminated sites in Khuzestan and were identified based on 16S rRNA gene sequencing. The catabolic genes were detected using PCR and hybridization analysis. Hydrocarbon degradation in liquid culture was evaluated by gas chromatography-mass spectrometry (GC-MS).. 23 indigenous oil-degrading bacterial strains were isolated from the wild oat rhizosphere, grown in severely oil contaminated soil in Khuzestan. Based on 16S rRNA gene sequence analyses, isolated strains were classified to Genera Acinetobacter, Pseudomonas, Enterobacter, Stenotrophomonas, Bacillus, Achromobacter, Ochrobactrum, Paenibacillus, Microbacterium, Curtobacterium and Sphingobacterium. Catabolic genes alkM, alkB and xylE, responsible for biodegradation of the alkanes and aromatic petrochemical compounds were detected in bacterial community inhabiting rhizosphere of the wild oat. The GC-MS analysis indicated that consortium of these bacteria was capable of reducing crude petroleum in the liquid culture by 40.5%, after 10 days. The results of the present study revealed the adaptability of microbes to the rhizospheric area and subsequently their great potential to be exploited for cleaning up hydrocarbon contaminated sites.. This study might be an important step towards the development of a phytoremediation strategy in the South of Iran..

The contamination of ecosystem components with petroleum and its derivatives is considered as one of the most crucial environmental threat in Iran، particularly in southern areas. Bioremediation has frequently been regarded as an appropriate and more practical alternative to clean-up petroleum hydrocarbons in the contaminated environments. Bioremediation optimizes conditions for microbial hydrocarbon degradation and uses the microorganisms and plants potential to metabolize contaminants resulting in their removal or attenuation in situ. This study aims at remedying an aged petroleum-contaminated soil using bioaugmentation and phytoremediation techniques. A consortium has been prepared using oil-degrading bacteria; 10% oil-contaminated soil was then inoculated with the consortium. Additionally، oat and/or barley were planted in certain treatments to separately evaluate the effects of plant-bacteria interaction on Total Petroleum Hydrocarbon (TPH) degradation and inoculum''s efficiency. TPH degradation value under unplanted and uninoculated conditions was only 2. 4% in the studied petroleum -contaminated soil after 4 months. However، the presence of the two plants elevated TPH degradation up to 30%، and bacterial inoculation resulted in only 20% TPH degradation. The significance of the plants in enhancing TPH degradation could be probably explained by promoting microbial populations، growth and activities. The highest amount of TPH degradation recorded was 44% and was observed with inoculated plants. The presence of plants in petroleum-contaminated soils promoted microbial populations and activities with increased microbial respiration and biomass well developed petroleum-degrading microbial population and decreased microbial metabolic quotient (qCO2)، hence، increased biodegrading of hydrocarbons.

Azotobacter chroococcum is an important PGPR (Plant Growth Promoting Rhizobacteria) producing compounds needed for plant growth. The aim of this research was to study the effects of different native strains of Azotobacter chroococcum on growth and yield of wheat under greenhouse counditions. Seeds of spring wheat (Triticum aestivum L. var. Pishtaz) were inoculated with some Azotobacter chroococcum strains capable of producing IAA, HCN, sidrophore and fixing molecular nitrogen. The inoculation of wheat with those strains had a positive, significant effect on biological yield, seed protein percentage, thousand seed weight, leaf area, N, P, Fe and Zn uptake, in particular, by wheat. The increased growth of wheat was most likely due to the production of IAA and enhanced nitrogen fixation by inoculated strains. Some strains of Azotobacter chroococcum native to Chaharmahal va Bakhtiari are established as PGPR. Results also support the efficiency of Azotobacter chroococcum as an important biofertilizer in wheat cropping systems. The selected strains had a significant effect on wheat growth and yield, including biological yield and seed quality under greenhouse counditions. This beneficial effect of Azotobacter chroococcum on wheat is attributed mainly to IAA production and, to some extent to non symbiotic nitrogen fixation in the rhizosphere. So, these strains can potentially be used to improve wheat nutrition of micronutrients such as Fe and Zn, in particular.