Symptoms Fusarium Oxysporum - Potato Iraq
Symptoms Fusarium Oxysporum - Potato Iraq
DOI: https://doi.org/10.56286/ntujavs.v2i2
Received: 07-08- 2023, Fusarium species are causing postharvest stem-end rot and dry rot throughout
Accepted: 19-10-2023, the potato growing season. At the current study, the causative wilt agent was
Published online: 28-12-2023
identified in potatoes which were grown in Nineveh province, northern of Iraq,
Corresponding author: in autumn season of 2020. The disease manifests through symptoms like
Name: Ali Hamood Thanoon partial yellowing between the veins on the leaves of potatoes or overall
Affiliation : Plant Protection wilting of the plant. Fungal species isolated from the infected potato
Department, Agriculture and plants were subsequently grown on potato dextrose agar, resulting in
Forestry College, Mosul developing aerial mycelia appeared as white along violet-dark
University, Mosul, Iraq. pigmentation. The fungus was identified from the phenotypic diagnosis as
Email:
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Fusariumoxysporum, and the pathogen was confirmed by Koch's hypotheses as
well as affirmed through molecular test identification, where methodology
Key Words: proved it. Molecular based pathogen identification was carried out through an
Potato, application of conserved DNA ribosomal region of internal transcribed spacer
Wilt. (ITS). Moreover, the complete verified ITS sequences are homologous to the
Solanumtubersum L., isolates of Fusariumoxysporum in GenBank database having 99% similarity. At
Fusariumoxysporum f. sp. Gen-Bank, isolated one in Iraq was given #: MH859948.1 being an Accession.
tuberosi, The best to the available knowledge, such is Fusariumoxysporum f. sp. tuberosi
1st molecular Potato record in Iraq.
©2023 NTU JOURNAL OF AGRICULTURAL AND VETERINARY SCIENCES, NORTHERN TECHNICAL UNIVERSITY.
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Corresponding author /NTU Journal of Agricultural and Veterinary Sciences (2023) 3 (4) : 199-204
2. Bead beater securing as fitted was done along a gently removed. In the EP’s horizontal unit, the
tube holder of 2 ml having association and process plate was fixed to a stand exemplified through the
at a speed as maximum for ≥ five min. utilized tank in EP. Moreover, TBE buffer filled the
3. In a microcentrifuge, Centrifugation was done tank while covering the gel surface.
for the Lysis Tube of ZR BashingBead™ at 10,000 Sample preparation
x g for one min. Following the mixing process, loading is processed
4. The 400 μl of supernatant was transferred to a to the gel pores. Three microliters of processor
Zymo-Spin™ IV Spin Filter (Orange Top) was loading buffer (Intron/Korea) and five liters of
carried out in collection tubes, after that fictitious DNA for EP (loading dye) were prepared.
centrifugation was performed at 7,000 x g for 1 Moreover, the electricity current of 7 V\C2 was
min. exposed for a time period of 1-2 hour as long as the
5. Further, 1,200 μl Fungal/Bacterial Binding movement of tincture took place to the other side of
Buffer of DNA was incorporated to the filtrate in gel. Thus, gel was further inspected through a UV
collection tubes from step # 4. source of 336 nm and after that gel was placed in
6. Mixture of 800 μl was transferred from step # 5 pool containing a staining solution of 500 ml DW
to a Zymo-Spin™ IIC Column 3in all collection and 3µl Red safe Nucleic acid.
tubes and then centrifugation was performed at ITC Gene Detection through PCR Utilizing
10,000 x g for one min. The ITS gene detection procedure is carried out
7. Flow discarding was done from tube of using amplification primers. Additionally, the ITS
collection and repeating to step # 6. segment was amplified using the ITS1 F: 5′-
8. To the column of Zymo-Spin™ IIC, DNApre- TCCGTAGGTGAACCTGCGG-3′ and ITS4 R: 5′
wash buffer addition of 200 μl was done in a new TCCTCCGCTTATTGATATGC-3′ forward and
tube of collection and centrifugation for one min at reverse primers. IDT (Integrated DNA
10,000 x g. Technologies Company, Canada) provided these
9. To the column of Zymo-Spin™ IIC, primer sets. Similar to that, PCR amplification was
Fungal/Bacterial DNA Wash Buffer addition of performed in a 25 µl total volume that contained
500 μlandcentrifugtion for one min at 10,000 x g. 1.5 µl DNA, 5 µl Taq PCR PreMix (Intron, Korea),
10. After moving the Zymo-SpinTM IIC column to and 1µl of every primer (10 pmol). After that
a clean 1.5 ml microcentrifuge tube, the column addition of DW into a 25 µl tube of a total volume.
matrix was immediately infused with 100 μl (at Thus, the cycling conditions as thermal were
least 35 μl) of DNA elution buffer. Therefore, a performed as following: Denaturation for three
10,000 x g centrifugation for 30 seconds was used minutes at 94 °C, after that 35 cycles for 45s
to elute the DNA. whereas, at 52°C, 94 °C, for 1 minute along with
For initial denaturation and annealing, the 72 °C for one minute. However, the final step
condition being optimal has recognized following incubation was done at 72 °C for seven minutes
preforming numerous experiments for gaining such with the help of a thermal Cycler (named as: Gene
condition, the temperature has changed during the Amp, PCR system 9700; Applied Bio-system).
Gradient PCR operation for all samples to choose Hence, PCR products were isolated from 1.5%
the best setting, and the DNA template agarose gel EP, further these were projected via
concentration has changed from 1.5-2 μl, which are UV exposure at 302 nm while following the
thought to be the two most important variables in process of red stain staining (Intron Korea).
primer annealing with complement.
Table 1. F. oxysporum isolate's RNA gene
DNA Agarose gel electrophoresis (EP) polymorphism
In order to assess the pieces of DNA, EP was Gene: 18S ribosomal RNA gene
performed following the procedure of extraction. No. Substituti Locati Nucleo Sequence Source Identit
As well as to perceive outcomes of PCR on type on tide ID with ies
interaction, where presence of standard DNA compare
distinguished PCR interaction result’s bundle size 1 Transvert 174 C\A ID: MH8 Fusariumo 99%
on the Agarose gel. ion 59948.1 xysporum f.
Agarose gel preparation Transvert 176 T\A sp. tuberosi
Based on (Sambrook et al ,1989),the agarose gel ion
was prepared in 1.5% condensation via agarose Sequencing and Sequence Alignment
melting of 1.5 g in formerly prepared TBE buffer Via 2% agarose gel EP, the products of PCR were
of 100 ml. Agarose was boiled then kept to cool at separated and envisioned through UV exposure at
(45-50 ᵒC). In the pour plate, the gel was poured (302 nm) following red stain staining or ethidium
where the agarose support plate has been prepared bromide.
following comb fixing to create holes that were Gene sequencing was done online biotechnology
holding the samples. Gently, the gel was poured to lab through (NICEM) website
avoid air bubbles formation and kept for thirty min (http://nicem.snu.ac.kr/main/?en_skin=index.html)
to cool. Of the agarose as solid, the comb was while machine used was Applied Biosystem DNA
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RESULTS
Isolation and Diagnosis
On PDA, the colonies of fungi were isolated and
comprised of aerial mycelia eing white in which
afterword formed pigments as dark violet which are
F. oxysporumcharacteristic (Figure 1, D). F.
oxysporumhas 3 conidia types: microconidia,
macroconidia, and chlamydospores. In contrast,
other Fusariumspp. have just 2 conidia types:
chlamydospora and macroconidia. Shapes of
microconidia, macroconidia, chlamydospores and
conidiophores (Figure 1, A-C) represent the
exclusive F. oxysporum characteristics. Fig 1. A, Conidiophores; B, Macroconidia and
microconidia; C, Chlamydospores; D, 1-week-old F.
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Corresponding author /NTU Journal of Agricultural and Veterinary Sciences (2023) 3 (4) : 199-204
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