Alternative methods to combat climate change effects of the negative impacts on Agriculture

1. Effects of mycorrhizal symbiosis on
alleviating drought stress, N.P.K.
absorption, physiological parameters
and yield of rice (Oryza sativa L.)
Spencer Poloma PhD/3
Supervisor: Dr. Maino
Co-supervisor: Prof A. Hartemink

2. Data Chapter 1
Morphological and Molecular Identification of
Abuscular Mycorrhiza Fungi (AMF)

3. 1. INTRODUCTION
• AMF research worldwide indicated
• AMF diversity (Rosendahl et al., 2009)
• Genetically complex even at single spore for genes (Stockinger et al., 2009,
Savary et al., 2018) and
• Proteins (Kuhn et al., 2001)
• Benefits to Agriculture and Environment
• AMF research in PNG –Nil
• OBJECTIVE:
• Isolate and identify indigenous mycorrhiza species

4. 2. MATERIALS AND METHODS
2.1 Table 1 Host plant sampling sites in the Morobe Province

5. Figure 1 Flow chart showing laboratory preparations for
morphological studies and subsequence molecular work.
Plant
Sample Sterilize
Culture
Fresh potato
broth PDA
Micros
-copy
Photo-
graph
DNA
Extraction
Soil
Sample
Spore
suspension
2.2 Laboratory pocedures

6. 2.3 Molecular methods
• DNA extractions
- with liquid nitrogen- DNeasy Plant Mini Kit, QIAGEN)
•PCR using pimer pairs –
•ITS1F/ITS4R
•GLOM1310F/GLOM5.8R
•Electrophoresis (after PCR)
•Gene sequencing and analysis

7. 3. RESULTS
3.1 Morphological characterization
Figure 2 Representative figures of AMF isolates on fresh potato broth dextrose agar

8. Table 2 Classification of 72 AMF isolates into morphotypes

9. 3.2 Molecular characterization
Figure 3 PCR amplicons by Generic primers, ITS1F/ITS4R (A) and Glomerale specific primers, GLOM1310F/GLOM5.8R (B)
displaying DNA fragments on 1 % agarose gel with a 1kb ladder (M) and negative control (C) for comparision. Generic primers
(A) displayed single bands of about 800 bp. Sample 30 was identified as Aspergillus japonicus. Glomerale specific primers (B)
indicated single bands of approximately 400 bp. DNA of isolate 30 in amplified with Glomerale specified primers was identified
as Glomus intraradices (Rhizophagus intraradices) and isolates 15 and 32 as Glomus irregulare (Rhizophagus irregularis) by
gene sequencing. Others produced species not of interest were discarded.

10. ISOLATE 15 Telemorphic form ISOLATE 32 Anamorphic form
Figure 8. Both isolates identified as R.irregularis indicating that AMF can switch
forms

11. Figure 4 Amplified sequence from isolate AMP001 (Sample 30).
Sequence processed by Macrogen, South Korea (nts: 1345
bps)
GGCCCGTAGCTCTGGTCCTTCGGGGCCACCTCCCACCCGTGCTTACCGTACCCTGTTGCTTCGGCGGGCCCGCCTTCGGGCGG
CCCGGGGCCTGCCCCCGGGACCGCGCCCGCCGGAGACCCCAATGGAACACTGTCTGAAAGCGTGCAGTCTGAGTCGATTGATA
CCAATCAGTCAAAACTTTCAACAATGGACTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAACTAATGTGAATTG
CAGAATTCAGTGAATCATCGAGTCTTTGAACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTCCGAGCGTCATTTC
TCCCCTCCAGCCCCGCTGGTTGTTGGGCCGCGCCCCCCCGGGGGCGGGCCTCGAGAGAAACGGCGGCACCGTCCGGTCCTCG
AGCGTATGGGGCTCTGTCACCCGCTCTATGGGCCCGGCCGGGGCTTGCCTCGACCCCCAATCTTCTCAGATTGACCTCGGATCA
GGTAGGGATACCCGCTGAACTTAAGCATATCACTAAGGGAAAAAGAAAAAACGGAGTGCTGGTCCTTGGGGCCTACCTCCACTA
TCTTACGTACCGGTGACGGCGACTTTCCTCTTAGTGGAGCTGTTTCTTCCTTTTATCTTCCCCGGAAACCCATGGAACATGTTGG
AAAACAGGCATTCAAAGTCAATTGATACCAATCATTCCAAACTTTCAACAGTGGGAATTTTTTGGGTTCCGGCCATCGTATAAAA
AAAACCCATCGCAAATTGCGGTTAAACAAAAGGTTGAAATGGCAAAAAATTTATTTGAAAACAACGCGGTCCCGTTTGGAACGC
CAAAATGGGGGCCCCCCCGGGGTTATTTCTCCGGGGGGGGGGACGGTGCCCCTTCCCAAAAACCCTTCTTTTTTCTCCCCCCC
CTCCCCCCCATCGGGGGTGGGGGGAGGGGGGCGGCGGCGCCCCCCCCCCCGGGGGGGGGGGGGGGCGCGGAGAAGAAAAA
AAAAAGGGGACCGCCCTCCCCTTCTCCGGTTCCCCGCCGAGAGGGAGAGGAGGGGGGGGGCCTTTTTCTTCACCCCCCCCCTC
TTTTTTGGGCGGGCCGCGGGGCGGGGGGGGTGTTGGGCCCCGCCCCCCCCCCCACTATTTTTTTTTTAAAAAAGAAGCGGCCT
CTCCGTGAAAAAAGGGGGGGGGTGGGGGGAGCCCCCCCCCCCCTTTATATAATTTTTTATCTCTTCTTTCTCTTTTCTGCGGCG
GCGAGAAAAAAAAAAGAAAAACGATGTAGATGGTAGCGTGGGACGGGTTGGTCCTGCCCCGCCGCGCGTGTGCGCTATTCTAT
TTATTTGGA

13. Figure 7 Sequence matches by Blastn analysis (n = 100). Same was done
for Glomerale Specific primers

14. AMF identified from rDNA amplicons of Glomerale specific
Primers and Generic primers.

15. (a) AMF can be cultured in-vitro and challenges
the world’s accepted view that AMF cannot be
cultured in-vitro.
(b) Therefore, further challenges the world’s
accepted view that AMF are obligate bio-
autotrophs.
Explanations for the progressive
outcomes:

16. Explanations for the progressive outcomes:
cont……..
(c) This study proved the above commonly accepted norms
wrong as the world’s widespread AMF species
Rhizophagus intraradices and R. irregularis were
cultured and identified by gene sequencing in this study
on fresh potato broth PDA implying that AMF are facultative
and not entirely obligate bio-autotrophs and can switch
form between and within species.
(d) As such, Aspergillus species at least under PNG climatic
conditions may behave like AMF and can switch form as
facultative fungi pocessing the genetic association
demonstrated in Fig.3., confirmed by genes sequenced.

17. Cont…….
•This findings confirms earlier work that criptic
genomic species exist within a species eg. R.
irregularis (Rosendahl et al., 2009) even multiple genes in
a species (Stockinger et al., 2009, Savary et al., 2018)
•multiple slightly differing variants of nuclear-
encoded ribosomal RNA genes (rDNA) found to be
present within a single spore (Redecker and Raab, 2006;
Lanfranco et al., 1999; Sanders et al., 1995).
•as well as multiple proteins genes in single spores
(Kuhn et al., 2001)

18. Conclusion
• Rhizophagus irregularis and R.intraradices are facultative and can be
cultured in the lab.
• Rhizophagus species are prevalent in all agroecosystems and crops
sampled
• Association of Rhizophagus species and Aspergillus species
genetically render flexibility to switch form morphologically or
genetically and can survive anywhere in the world making
Rhizophgus intraradices and R. irregularis the most widespread
worldwide species with such genetic nature
• These results challenges the world’s accepted view that AMF are
obligate bio-autotrophs and cannot be cultured and proposed that:
• World’s predominant species of AMF are facultative-can be cultured
and forms the basis for detail AMF research in PNG.

19. Recomendations
Sample plant species across Papua New Guinea to
study AMF diversity and:
1. Further study the genetic association of AMF
and other fungi species
2. Further study culture conditions required for
longer terms in-vitro maintainance.

20. What To Do Next:
(a) Complete and submit paper 1 on “Isolation and Identification
of AMF species in PNG” and prepare others
(b) Evaluation of isolated AMF in Pot Trials under optimum and
drought simulated glasshouse conditions
(c) Analyse results and finalise Thesis

21. THE END