Akademik Veri Yönetim Sistemi
UFUK 2020 Projesi, 2019 - 2021
Salinization of soil
is a serious problem and is increasing steadily in many parts of the world, in
particular in arid and semiarid areas. Saline soils occupy 7% of the earth’s
land surface and increased salinization of arable land will result in to 50%
land loss by the middle of the 21st century. This project was establish to: a)
Isolation and enumeration of AMF spore populations in soils of different
sampling sites showing a gradient of salinity, b) Determination of Mycorrhizal
colonization indices on different host plants in sampling saline sites, c)
Relationship among different soil physico-chemical features and AMF spore
population and mycorrhizal colonization indices, d) Diversity and community
composition assessment of AMF species in different sampling sites based on
morphological and molecular methods and f) In vitro mass propagation of
AMF prevalent species in laboratory conditions. For this purpose, Soil and root
samples were collected from different regions including Ankara, Eskisehir,
Igdir, Samsun, Izmir and Konya. From 75 collected soil samples, 28 samples
selected based on five EC groups. Different physicochemical parameters were
measured in all soil samples. Significant differences observed among soil
samples EC in sampling regions. 24 different host plants in all sampling
regions were also identified, dried and kept in institute herbarium.
Significant difference observed in spore density among soil samples in
different regions based on host plant species as well as soil parameters. The
minimum and maximum average of spore density observed in Konya region from the Suaeda
maritima and in Eskisehir region from wheat rhizospheric soil,
respectively. The Eskisehir soils had the highest spore numbers and Samsun had
the least spore numbers mean among the sampling regions. Host plant roots
cleared and stained for observation mycorrhizal colonization as well as
colonization indices assays (F% and M%). Fungal structures including mycelia,
vesicles and arbuscules observed in all root samples. F% index had no
significant difference among soil samples but the M% index were different
except of Samsun soil samples. Three way ANOVA showed the significant effects
of sampling regions, soil parameters as well as host plant species on
Mycorrhizal colonization (M%). The highest and the least total averages of
spore numbers, F% and M% obtained in samples collected from Eskisehir and
Samsun regions, respectively. So, it seems that the most fungal spore numbers
in the samples means the most values of mycorrhizal colonization. Pearson
correlation analysis was also confirmed the positive significant correlation
between spore density and root colonization index (M%). For morphological
identification of AMF species, trap culture was established using white clover
as host plant in greenhouse condition for 120 days. Then, microscopic slides
prepared from isolated AMF spores. Species identification was carried out based
on spore morphology and wall characteristics using standard manuals and
specialized websites. Then, different biodiversity indices were measured.
Totally, 33 definite and 1 undefinite AMF species were identified from 12
genera, 6 families and 4 orders. The diversity of fungal genera as well as
species was heterogenous among soil samples. It means that some fungal genera
or species only observed in specific regions or samples. Also, the dominant AMF
genus was Glomus with the most identified species. Samples from
Eskishehir and Samsun had the most and the least species richness values,
respectively which is in agreement of the results of Spore density and colonization
indices. On the other hand, R. intraradices determined as dominant
fungal species with the highest values of FO, RA and IV indices. Gigaspora
gigantea found as the rarest fungal species in all sampling regions due to
the least IV value. Jaccard index showed the most similarity of AMF
species between Eskishehir-Konya regions (0.74) as well as Ankara-Igdir regions
(0.74). Also, the least similarity observed between Ankara-Izmir regions
(0.34). Species richness (SR) mean ranged between 5.33 in Samsun to 14.67
in Eskishehir. Shannon index (H) average varied between 0.99 in Samsun
to 1.76 in Eskishehir. Comparatively to Hmax, Eskishehir and Konya
regions presented the higher AMF biodiversity. The Eveness index ranged between
0.73 in Samsun to 0.98 in Eskishehir. High Equitability or Eveness values
obtained in Eskishehir and Konya regions (close to 1) testified to an equitable
distribution of AMF species. Lowest E value in Samsun region referred to
the presence of some rare species, which were unequal distributed. The
ecological conditions of Eskishehir and Konya regions seemed to be favorable
for all AMF identified species and offered equal survival opportunity. Results of
correlation between important soil parameters with AMF biodiversity parameters
showed that soil EC, available P and K had negative effects on all of AMF
biodiversity indices. Also, soil texture is an important factor in AMF
biodiversity. Soil organic matter had most significant positive effects on
fungal species richness, Shannon index as well as Eveness index in different
sampling regions. Results of carrot transformed hairy roots showed that two
kinds of roots started forming on the carrot discs after 8-10 days. The first
type was delicate, aerial hairy roots without any growth after cutting from the
discs and rotted after one week. In contrast, the second type was slightly
thicker transformed roots. One desirable characteristic of these transformed roots
was their ability to quickly form numerous lateral roots. Another
characteristic observed in transformed roots was the inversion of their
geotropic habit of growth. Roots were initiated from the both sides of the
discs. Hairy root initiation continued to occur from 8-10 days to 3-4 weeks. For
In vitro propagation of AM fungi, we used two systems: Root organ
culture system using the transformed hairy roots and AM-Plant in vitro
culture system by sandwich methods. The fungal spores, sporocarps as well as AM
colonized root fragments were selected as propagules. The fungal propagules
were disinfected using the treatments consisting of Chloramine T with
antibiotics. After disinfection process, only fungal spores and sporocarps
could be germinated without any other infection and used for further
co-cultivation process. In our study, the spores of Rhizophagus intraradices
and sporocarps of Funneliformis mosseae were germinated after 7 and 19
days followed by disinfection and culturing on MSR medium without any infection.
In root organ in vitro culture system, after initial contact between the
germ tube and the transformed root, the intercellular colonization took place
around the 12th and 20th day in
R. Intraradices and F. Mosseae, Respectively. During next 18-20
days, multiply laterial branches on the root and media surface by extensive
hyphal proliferation could be observed. After that, rapid enlargement of
mycelium in the form of clusters and the formatiom of new spores was observed
within 30 to 45 days. In sandwich method, we used the
sterilization/scarification of Medicago sativa (alfalfa) seeds using
sodium hypochlorite followed by placing the seeds in Petri plates containing
40ml MSR medium without sucrose and vitamins. The seeds were germinated within
3-5 days. Two AM fungal species, Rhizophagus fasciculatus and Claroideoglomus
etunicatum were propagated by this system. The life cycle of fungi in this
method was slightly different from root organ culture method. The spores of R.
fasciculatus and C. etunicatum were germinated 3-5 days and 5-7 days
after inoculation, respectively. The pattern and intensity of spores were
similar to
in vitro culture studies using transformed roots. The growth of
germinating hyphae in both species in order to find the host roots and thus
make the first contact with them were much faster than root organ culture
method. The germinating hyphae were able to establish intitial contact with
host roots after 24-48 hours. The growth and development of the extra radical
mycelia networks were strarted 2-3 days after the first contact between the
fungus and the host root and continued until the end of the study period (1
month). Development of hyphal networks in R. fasciculatus was very
extensive and often 13-12 days after the establishment of symbiotic association
between the fungus and the host roots, the extra radical mycelia were spread in
all the plate. The first spores were also observed 20 days after inoculation.
In the case of species C. etunicatum, the first spores were formed 13
days after inoculation. These spores were morphologically similar to the
inoculated spores and were approximately 85 μm in diameter. Soil and root
samples DNA was extracted in triplicate using DNA extraction kits. All DNA
samples were stored at −20°C until PCR amplification. DNA was extracted
successfully from all root and soil samples with A260/280 ratio of 1.8-1.97 as
well as more than 100ng/µl DNA which are good values for quantity and quality
of extracted DNA for PCR amplification. Based on reviewing all the molecular
studies on AMF, we decided select sequences from small subunit (SSU) of rDNA
region for Nested PCR-DGGE amplification. In addition, based on literatures, we
selected the AM1, NS31, Glo1 and
NS31-GC primers for PCR amplification. The nested PCR was carried out in two
rounds. The first PCR was done with AM1/NS31 primers and the second PCR was
done with Glo1/NS31-GC primers. AMF 18S rDNA fragments of approx. 195 bp were
successfully amplified from samples by the nested PCR approach. The Nested PCR
product (20 µL) was subsequently analyzed by DGGE system. The gels containing
6.5% acrylamide were poured with a gradient of 35%–55% denaturant. The gels
were then stained with SYBR Green, illuminated with ultraviolet light and
digitally photographed. The observed bands exhibited good resolution, visible
intensity and distinct DGGE fingerprints characteristics with different
intensities indicating a different AM fungal community structure in each
sample. Based on the analysis of DGGE profiles, it was clearly seen that the
AMF communities in the soils were different from those in the corresponding
plant roots. For fingerprinting analysis, the DGGE patterens with distinctive
bands were selected. Totally, 21 samples including 12 soils and 9 root samples
were analyzed. For determination of ecosystem diversity, we used the species
richness (SR) and Shannon-Wiener index (Hʹ) in soil and root
samples of each sampling regions. Similarities between DGGE patterns were
calculated by generating the dendrogram utilizing the clustering method UPGMA. The
species richness (SR) and the Shannon-Wiener index (Hʹ) of AMF
communities showed remarkable differences among the six sampling regions in the
soil and root samples. It could be concluded that the most and the least species
richness and diversity observed in soil and root samples from Eskişehir and
Samsun regions, respectively. The results obtained by DGGE fingerprinting
analysis is in accordance with the results obtained by morphological analysis. AMF
community composition by cluster analysis of DGGE profiles showed two main
clusters. The first cluster included IZ1-2 and IZ1-5 soil and root samples. The
second cluster was divided in two main subclusters including one Izmir soil
sample (IZ3-1) as a separate group and all the soil and root samples from
Eskişehir, Konya, Samsun, Igdir and Ankara regions. The samples from Eskişehir
and Konya clustered together in subcluster 1. The results showed that the
subcluster 2 was divided in 2 more subclusters. The samples from Konya and Igdir
put together in one subcluster. In addition, the soil samples collected from
Ankara consisted another subcluster. The result of this clustering was in
accordance with the results obtained from Jaccard similarity index of AMF
species in sampling regions. Single dominating DGGE bands (21 bands, M1-M21) were
excised from the acrylamide gel for further phylogenetic studies. DNA was eluted
and amplified with primer pair NS31/Glol. The PCR products were recovered on 1%
agarose gel, purified, cloned with pGEM-T Easy vector followed by transformation
into Escherichia coli DH5α competent cells. The positive clonal plasmids
were sent to BM Yazilim Danis. ve Lab. Sis. Ltd. Şti, Anakar-Turkey for
sequencing. Sequences from DGGE bands were edited in MEGA 6.0 and their
similarities were determined using the BLASTn program. The sequences were
multiple aligned with the sequences from major clades of Glomeromycota with
more than 95% homology using the CLUSTALX program. The neighbor-joining (NJ)
algorithm was used to construct phylogenetic relationship among DNA sequences
using MEGA 6.0 with bootstrap values of 1000 replicates. The DNA sequence
fragment lengths of all 21 bands were approximately 200 bp. The results of the
BLAST showed that all the sequences had high similarity
(greater than 97%) to the sequences from members of Glomeromycota. The M2, M3,
M6 and M17 bands had the highest similarity (100%) with the Funneliformis
caledonium, F. mosseae, uncultured Pacispora and Paraglomus
occultum species, respectively. The M1, M4, M7, M12, M14, M15, M19 and M21
bands were belonged to Rhizophagus fasciculatus, Rhizophagus intraradices,
Funneliformis constrictum, Gigaspora margarita, Acaulospora spinosa,
Acaulospora koskei, Claroideoglomus claroideum and Archaeospora trappei
species, respectively with 99% similarity. The M5, M9, M10, M11, M13, M16 and
M18 bands had 98% similarity with Funneliformis geosporum, Gigaspora
gigantea, Acaulospora laevis, Gigaspora margarita, Gigaspora decipiens, Glomus
versiforme and Glomus macrocarpum species, respectively. The M8 band
had high similarity with Rhizophagus clarus (98%) and Rhizophagus
manihotis (99%), respectively. Also, the M20 band had the same similarities
with Claroideoglomus lamellosum (98%) and Claroideoglomus etunicatum
(99%), respectively. It is supposed that other primers for 18SrDNA, 28SrDNA or
ITS regions could be used for discrimination of these bands. All the DGGE bands
(M1-M21) belonged to 9 AMF genera including Rhizophagus, Funneliformis,
Pacispora, Gigaspora, Acaulospora, Glomus, Paraglomus, Claroideoglomus and Archaeospora;
17 definite and 1 indefinite species (uncultured Pacispora) and 4
uncertain species (Rhizophagus clarus, Rhizophagus manihotis,
Claroideoglomus lamellosum, Claroideoglomus etuniactum). We compared the
results obtained by molecular analysis with morphological identification. The
results showed that some species could not be identified by molecular analysis.
Also, regarding the Paraglomus genus, in morphological analysis the P.
bolivianum was identified while, in molecular analysis the P. occultum
was obtained. It is supposed that needed for further studies to exact
identification of fungal species. On the other hand, in molecular studies the Pacispora
robigina could not be identified and the band was similar to sequences
belonged to uncultured Pacispora. In morphological studies, the Archaeospora
genus was identified but without any defined species, while, in molecular
analysis it was concluded that the species is Archaeospora trappei.