Rajan L Fradlin Singh

M.Sc Agricultural Microbiology

 Introduction

 Pink Pigmented Facultative Methylotrophs, commonly

abbreviated to PPFMs, are bacteria that are members of the
genus Methylobacterium .
 Commonly found in soil, dust, various fresh water supplies
and on plant surfaces.
 Their pigmentation, which is frequently pink but may also be
yellow or orange.
 This color is present due to the carotenoid pigments within
the cell.
Contd
 Pink pigmented facultative methylotrophs (PPFMs) are diversified
group of microorganisms that promote plant growth by producing
indole acetic acid (IAA) and cytokinins.
 Pink pigmented facultative methylotrophs (PPFMs) are the prime
inhabitants of phyllosphere region of wide variety of plant species.
 Based on the plant-microbe interaction Methylobacterium sp. are
classified as biostimulator, biofertilizer and biocontroller as they
directly influence the plant growth by producing phytohormones
especially auxins.
Genus:Methylobacterium
 Microbes Involved in PPFM

 Methylobacterium adhaesivum
 M.aminovorans
 M.aquaticum
 M.chloromethanicum
 M.dichloromethanicum
 M.extorquens
 M.fujisawaense
 M.hispanicum
 M.isbiliense
 M.lusitanum
 M.mesophilicum
 M.nodulans
 M.organophilum
 M.podarium
 M.populi
 M.rhodesianum
 M.bullatum
M.rhodesianum M.rhodinum
Methylobacterium populi Methylobacterium marchantiae
M.hispanicum M.bullatum
Methylobacterium phyllospherae
 Characterization of PPFM
 Methylobacterium spp. are aerobic,facultatively
methylotrophic,fastidious, slow-growing bacteria.

 Size:Small (1 to 2mm in diameter), pink-pigmented

colonies on ordinary solid media.

 Temperature:Optimum temperaturen 25 and 30°C

,moderate growth at 35°C and no growth at 42°C.
Contd
 Shape:Gram-negative pleomorphic, non-spore-
forming,vacuolated, rod-shaped cells and have one
polar flagellum.
 Medium for PPFM
 Tripticase soy agar, sheep blood agar, nutrient agar,and
Mueller-Hinton agar and on plate count agar and
Reasonenr’s 2A agar.

 The best growth was observed on Sabouraud dextrose agar

and buffered charcoal yeast extract agar.

 Two media used for plate count analysis in drinking water.

 The commonly used medium for PPFM is AMS Medium.

 Composition of AMS medium

K2HPO4(Dipottasium Phosphate) 0.7 g

KH2PO4(Pottasium Di Hydrogen Phosphate) 0.54 g
MgSO4(Magnesium sulfate) 1.0 g
CaCl2(Calcium chloride) 0.2 g
FeSO4(Ferrous Sulfate) 4.0 mg
NH4Cl(Ammonium chloride) 0.5 g
ZnSO4(Zinc sulfate) 100.0 mcg
MnCl2(Magnesium chlroide) 30.0 mcg
H3BO3(Boric acid) 300.0 mcg
CoCl2(Cobalt chloride) 200.0 mcg
CuCl2(Copper chloride) 10.0 mcg
NiCl2(Nickel chloride) 20.0 mcg
Na2MoO4(Sodium molybdate) 60.0 mcg
Distilled Water 1 litre
PPFM on AMS medium
 Cytokinin Production
Bacterial isolates were grown in ammonium mineral salts
medium.

The culture broth was centrifuged at 10000 rpm for 15 min to

pellet the cells.

The cell-free culture suspension was acidified to pH 2.9 with

0.1N HCl and extracted thrice with equal volumes of diethyl
ether to remove auxins.

The pH of the aqueous phase was adjusted to 7.0.

Contd

The residue was dissolved in 3 ml of HPLC grade

methanol.

The methanol fraction was filtered through 0.2 μm

bacterial
filter and used for high performance liquid
chromatograph analysis.
 IAA production
AMS broth added with L- tryptophan (100 mg l-1) was prepared.

100 ml quantities were dispensed in 250 ml Erlenmeyer flasks.

The flasks were sterilized at 15 psi for 20 min.

One ml of the inoculum (109 cfu m1-1) of PPFM isolate was

added and incubated at room temperature in a shaker for 10
days.
Contd
The flasks were wrapped with black paper during incubation to avoid
photo inactivation.

The culture broth was centrifuged at 10000 rpm for 15 min and the
supernatant was taken.

The pH of the supernatant was adjusted to 2.8.

Extraction was done thrice using equal volumes of diethyl ether at 40C.

Organic fractions were pooled and evaporated in dark. The residue was
dissolved in 2 ml methanol.
 Gibberellic acid production
AMS broth was prepared and 50 ml quantities were
dispensed in 250 ml Erlenmeyer flasks.

The flasks were sterilized at 15 psi for 20 min.

One ml of the inoculum of PPFM isolates with a

population density of 109 cfu m1-1 was added to each
flask and incubated for 10 days.

The culture broth was centrifuged for 15 min at 10,000

rpm and supernatant was taken.
Contd
The cell pellet was re-extracted with phosphate buffer
(pH 8.0) and again centrifuged.

Both supernatants were pooled, acidified at pH 2.5

using 0.1N HCl and partitioned with equal volumes of
ethyl acetate for five times.

The ethyl acetate phase was dried at 32°C and the

residue redissolved in 15 ml of methanol.
 Bioremediation using PPFM
 Methylobacterium spp. are able to biodegrade a variety of
organic toxic compounds.

 Van Aken et al., observed that Methylobacterium sp. strain

BJ001 in pure culture was able to degrade several toxic
explosives.

 The explosives are Trinitrotoluene (TNT), Research

Development explosive(RDX) and High Melting Explosive
(HMX).
 M. extorquens DM4 also has the ability to degrade a volatile
and toxic halogenated solvent dichloromethane which is
mainly used and produced by industry.

 The genera of Methylobacterium is identified that it

bioremidiate contaminate water and heavy metals in soil.

 The potential of the methylotrophic genera is shown by their

tolerance to high doses of several heavy metals, such as nickel
(Ni), cadmium,cobalt,zinc,arsenic,lead and mercury.
 Biotechnological uses of Methylobacterium

 Methylotrophic bacteria can produce several industrial

products and biodegradable compounds.
 Methylobacterium spp., are able to produce highly
resistant biodegradable plastic that is similar to
conventional plastic.
 Examples of such biopolymers are the biodegradable
polyesters polyhydroxybutyric acid (PHB) and
polyhydroxyalkanoate (PHA).
 Methylobacterium spp. are able to produce is
glyoxylate.
 Verginer et al., reported that M. extorquens DSM
21961 in vitro can increase the production of two
furanoid compounds, 2,5-dimethyl-4-hydroxy-2H-
furanone (DMHF) and 2,5-dimethyl-4-methoxy-2H-
furanone, which are responsible for strawberry flavor,
showing that the bacterium can influence fruit quality.
 Inhibition Of Plant Pathogens

 Methylobacterium spp., can protect host plants by the synthesis of

a large spectrum of antimicrobial molecules nutrient competition
with pathogens or by inducing systemic resistance.
 Seed treatment with Methylobacterium sp. induced significant
protection against Aspergillus niger and Sclerotium
rolfsii pathogens in groundnut.
 Methylobacterium sp. IMBG290 inoculum was able to induce
potato resistance against Pectobacterium atrosepticum by
activating the plant antioxidant system.
 Ardanov et al., evaluated the ability
of Methylobacterium sp. IMBG290 to induce resistance
in several potato cultivars against P.atrosepticum,
Phytophthorainfestans, and Pseudomonas syringae pv.
Tomato.
 Yim et al., reported the induction of defense responses
in tomato with Ralstonia solanacearum after treatment
with four different Methylobacterium strains.
 Drought Mitigation
 PPFM was found superior in improving stress tolerant index
and catalase enzyme activity.

 It can protect the plant under abiotic stress condition.

 Foliar spray of PPFM was found to superior in improving

relative water content ultimately improve the drought tolerant
capacity in tomato.
 PPFM excrete plant growth hormones auxins and
cytokinins that influence germination and root growth
and help plants to endure water stress.

 PPFM enhances seed germination during stress

conditions
Rice field before and after application of ppfm
 Mass Production of PPFM
Preparation of modified medium for faster growth of PPFM:

 The PPFM is generally cultured in Ammonia Mineral Salt

(AMS) medium. Since the AMS media supports slow growth
of PPFM, a modified glycerol peptone medium with 0.5 per
cent methanol was developed to enhance the growth rapidly
within 24-48hrs.

 To avoid fungal contamination an antifungal compound

(cyclohexamide - 2ppm) was incorporated in the medium.
 The modified glycerol and peptone medium prepared in
fermentor .The modified Glycerol and Peptone medium
was filled in fermentor .The sterilized broth in the
fermentor was cooled down to 85 C temperature at zero
pressure before collecting it in plastic cans.

 Plastic container used for media collection upto 3/4th

the total volume for culturing of PPFM .The broth was

collected directly from the fermentor.

 After this, the container was closed air tight with outer

plastic cap and allowed to cool completely for 12 to

18hours.
 PPFM mother culture was inoculated to the cooled modified
medium upto a concentration of 7 percent.
 Finally, the cans were closed with outer lid and a small nail
hole was made in the middle of the outer cap.
 This provision of holes made on the inner and outer plastic
caps facilitated the release of carbon dioxide produced
during multiplication of cells.
 The PPFM inoculated cans were labeled, stored at room
temperature for 24-48h.
 Application of PPFM
 Seed treatment-Imbibe seed in 0.1%
volume for 5-10 min.
 Foliar spray of 1% PPFM.
 Spray at critical stage of crop
growth(or)30 days interval.
 Don’t mix with pesticide/fungicide.
 Effect of PPFM on growth and yield crops
 Fast seed germination and seedling growth.
 Accelerate vegetative growth.
 Increase leaf area index and chlorophyll content.
 Improves fruit quality colour and seed weight.
 Yield increases by 10%.
 Mitigate drought.
 Foliar spray of 2% PPFM on tomato at 25 and 45 days after
transplanting maintained the leaf water potential and leaf
temperature under drought .
 Reduced flower drop percentage and increased the fruit
yield .

 PPFM excrete plant growth hormones auxin and

cytokinin that influence germination and root growth.

 PPFM helps plants to endure water stress.

 Improves earlinesss in flowering,fruit set and

maturation.
 PPFM increases fruit quality,color and seed weight.

 M. oryzae was able to increase Cd and Ni tolerance in

tomato plants by decreasing Cd and Ni uptake and
promoting plant growth.
 Conclusion
 PPFM are generally the species bacteria which comes
under the family Methylobacterium.

 They can be mass multiplied and applied to the field.

 By applying PPFM yield of fruit and seed quality is

increased.

 It plays a major role in drought mitigation in rice crops.

 Farmers can easily benefited by this microorganism as
it plays a wide variety of role in the field.

 These M-trophs are the future of agriculture.