Role of Phylloplane bacteria in plant disease management

1. Phylloplane Bacteria
&
Their Role in Plant Disease Management
PLPATH-603
Advances in Plant Pathogenic Prokaryotes
Presented By:
Niyaj Ahamad
Id. No. A1163/19/22
Ph.D. Agril. Biotech.
1st Year/1st Sem.
Presented To:
Dr. Manoj Kumar Chitara
Department of Plant Pathology
Acharya Narendra Deva University of Agriculture & Technology
Kumarganj, Ayodhya-224229 (U.P.)

2. Introduction
Physllosphere are the entire aerial habitat and phylloplane defined as leaf surface area.
Plant surface associated microbes are known as epiphytes while they reside inside are known as
endophytes.
Phylloplane comprise of diverse microbial communities that inhabitate the leaves include bacteria, fungi,
algae, yeasts and nematodes
Plant tissues, leaf surface i.e. leaf exudates supply nutrition, moisture, pH and temp. to the phylloplane
microbes for their survival.
Plant and phylloplane interaction leads to the growth, development and protection of plants.
Pathogenic fungal spores accommodated on leaves while exposed to continuous air current and trap on
waxy surfaces and tricomes
Phyllosphere dominating microbes include Methylobacterium, Sphingomonas, Pseudomonas while
fungal communities include members of Ascomycota and Basidiomycota they contain antagonistic
activities against phytopathogens.
Phylloplane microbes have been explored and found to play an important role in antimicrobial (i.e.
antibacterial and antifungal) activities against phytopathogens.

3. Antimicrobial compounds secreted by phylloplane microfungi on aerial surfaces can be directly detrimental to
pathogens or via induction of systemic acquired resistance (SAR) in the plant cells.
Phylloplane microbiota promotes plant growth through the production of phytohormones e.g., indole acetic
acid (IAA), cytokinins, etc. Sphingomonas spp. produces plant growth stimulating factors i.e., IAA which
suggest that plant hormones produced by phyllospheric bacteria enhance plant growth.
Microorganisms are also known to play an important role in global processes like nitrogen fixation,
nitrification and phosphate solubilization.
Phylloplane microfungal metabolites have enhanced the activity of Ribulose-1,5-bisphosphate carboxylase/
oxygenase (Rubisco) thus playing an important role in the photosynthetic process.
Siderophores produced by iron competing bacteria, antibiotics such as DAPG and pyocyanin, biosurfactants
such as 2R, 3R-butanediol produced by B. subtilis GBO3 (130) and a C13 volatile emitted by Paenibacillus
polymyxa are chemical metabolites which can protect plants (Pieterse et al. 2014).
Phylloplane fungi such as Cladasporium spp., Penicillium spp., and Aspergillus flavus could inhibit mycelial
growth and spore germination of Helminthosporium oryzae thus preventing rice brown spot Harish et al.
(2007).
The metabolites of Trichoderma viride and Aspergillus flavus were found to be effective in inhibiting the
pathogenicity of Alternaria brassicae against rabi crops (Yadav et al. 2011).

4. Role of Phylloplane Microbes in Systemic Acquired
Resistance (SAR) and Induced Systemic Resistance (ISR)
SAR is activated throughout a plant by exposure to elicitors from virulent, avirulent or
nonpathogenic microbes or artificial stimuli while ISR is the resistance mechanism in plants
which become activated on infection due to an invading pathogen (Kamle et al. 2020).
Nonpathogenic F. oxysporum could induce systemic resistance and defence responses
against pathogenic F. oxysporum f. sp. asparagi in Asparagus officinali (He et al. 2002).
Trichoderma produce a toxic compound which had antimicrobial activity against pathogens
as well as secreting compounds that stimulated the plant to produce its own defense
metabolites.
Buxdorf et al. (2013) reported that local inoculation of Pseudozyma aphidis elicited
induced resistance in Arabidopsis and reduced growth of Botrytis cinerea on local and
systemic leaves.

5. Microbial and bioactive soil amendments for improving strawberry crop growth, health, and fruit yields: a 2017–2018
study (Dara 2019).

6. Phyllospheric microorganisms play crucial roles in plant growth and thus provide ecosystem services like
carbon (C) sequestration, nitrogen (N) fixation and bioremediation, thereby enhancing crop yield and
improving soil health.
Several phylloplane inhabiting microbes produce phytohormones such as auxin, gibberellic acids, and
cytokines and could fix nitrogen and mobilize nutrients (Dourado et al. 2015).
Indole acetic acid is also produced by phyllospheric microorganisms which stimulates root growth and
eventually enhances root contact with soil and increases nutrient uptake.
Due to this ability, such phylloplane microbial inoculants as: Bacillus, Microbacterium, Acinetobacter,
Proteus, Psychrobacter, Pseudomonas, etc., are now recommended as substitutes to chemical fertilisers
(Batool et al. 2016; Mohanty et al. 2016). ulgarelli et al. 2013).
Succinate dehydrogenase (SDH) produced by Pseudomonas syringae exhibited reduced SDH activity
(Mitra et al. 2013) which located in the inner membrane of mitochondria influences photosynthesis,
induces fungal defence responses in plants and controls stomatal functions and root elongation (Huang and
Millar 2013). succinate oxidation to fumarate by CO2 leads to inhibition of SDH leading to succinate
accumulation which is toxic to plant tissues due to anomalies in SDH lead to reduction of mitochondrial
H2O2 production and to an increase in host susceptibility against pathogens (O’Brien et al. 2012).

7. Factors affecting phylloplane microorganisms
General
Microorganism populations of the leaf surface are affected by abiotic factors such as the
microclimate and biotic factors such as the leaf surface itself and interactions between the
microorganisms.
Abiotic factors
1. Microclimate
The boundary layer of a leaf is the layer of air immediately surrounding the leaf, between the
ambient wind speed and the surface of the leaf where the wind speed is nil. The thickness of
the boundary layer is determined by the presence or absence and density of trichomes
(Mauseth 1988), and is usually less than 1 mm. This layer is sometimes referred to as the
phyllosphere, and it is this layer in which phylloplane microorganism interactions take place.
The microclimate in the boundary layer of the leaf usually differs considerably from the
ambient climate (Dix and Webster 1995).

8. 2. Temperature
The temperature of a leaf depends on a number of factors such as the ambient temperature and the solar
irradiation, as well as position, shape, surface topography, transpiration, wind speed and wetness of the
leaf. Temperatures can vary over the surface of a single leaf but generally by not more than 2-3 °C, with
the highest temperature occurring at the center of the leaf. Leaves are often cooler at night and warmer
during the day than the surrounding air, and the temperature of the leaf surface fluctuates more in the
periphery than within the canopy.
3. Humidity and leaf wetness
Under normal conditions, leaves transpire continuously, causing the humidity of the boundary layer to be
higher than the surrounding air, depending on the thickness of the boundary layer, the number and status
of stomatal apertures and the availability of water.
Due to a generally greater number of stomata and a lower occurrence of convection currents, the
humidity is often higher on the abaxial surface than on the adaxial surface. Furthermore, a lower solar
irradiation on leaves within the canopy, results in a lower day temperature and less fluctuation in
humidity than in the periphery.
During most nights, dew forms on the leaves due to a lower leaf temperature than the ambient
temperature, and this is a very important factor because most phylloplane microorganisms can only
germinate and grow on wet leaf surfaces or when the relative humidity exceeds 95%.

9. 4. UV light
Natural ultraviolet (UV) radiation can be harmful and often lethal to many microorganisms because it
"damages DNA by causing adjacent pyrimidine bases to join up as dimers and by causing a number of
other subtle changes“.
Some pathogenic fungi that produce long germ tubes are therefore less likely to survive on leaf surfaces
than fungi that produce short germ tubes or pigmented haustoria immediately after germination.
5. Wind
The wind speed within the boundary layer of the leaf is low relative to the ambient wind speed. However,
many microorganism propagules are windborne and therefore many spores form on top of conidiophores,
which expose them to higher wind speeds outside the boundary layer.
Deposition of airborne fungal spores onto the leaf surface occurs by wind impaction, for which the spores
need to pass through the boundary layer, and air turbulence. Large spores are more easily deposited than
small spores, because of their more favorable surface areito-mass ratio.
6. Nutrients
Various types of debris, such as pollen, honeydew, fungal spores and other substances on the leaf surface,
provide nutrients for the survival and growth of phylloplane microorganisms and have a major influence
on microbial diversity and activity.
Pollen grain release sugars, amino acids and proteins, which can be utilized by germinating spores.

10. 7. Pesticides
The use of foliar pesticides to control diseases can cause major disruption of phylloplane microorganism
populations, often reducing the number and diversity of organisms. This can have a negative effect on
naturally-occurring biological control, which in some cases, makes the plants more susceptible to other
disorders.
8. Pollution
Phylloplane microorganism populations are often affected by atmospheric pollution in the form of lead and
sulphur dioxide emissions and also by ozone and oxides of nitrogen. Plant damage may be caused by the
direct effect of high concentrations of toxic material, but also indirectly by reducing the activity of
sensitive phylloplane microorganisms.
Biotic factors
1. Leaf position
Many plant species maximize their ability to photosynthesize by heliotropism. Sunflower and bean leaves
were reported to face north to north-east during the morning, disperse during the day and face west during
the evening, while leaves of many deciduous woody plants were found to be horizontally oriented in shade,
but vertically oriented during the middle of the day.
Heliotropism ensures optimum solar irradiation, resulting in generally higher temperatures and UV light,
and lower relative humidity of the leaf surface than the surrounding air, conditions generally considered to
be inhibitory to the development of microorganisms.

11. 2. Leaf topography
The distribution and shape of leaf surface features such as veins, trichomes, stomatal pores, glands,
epidermal cells and epicuticular wax crystals determine the topography of plant leaves. The topography of
plant leaves varies among different plant species, among different leaf ages of the same species, and for
different locations within plants. Topography may also differ for leaves with different functions as well as
between the abaxial surface, which generally has more trichomes and stomata and the adaxial surface of the
same leaf.
3. Surface wax
The adaxial leaf surface generally has a thicker cuticle with more waxes than the abaxial surface, which
acts to prevent transcuticular transpiration and to deflect excessive sunlight as waxes can contain anti-
microbial substances.
4. Leakage from leaf
Substances from within the leaf leak continuously into water films on the leaf surface. These water films
may originate from dew, rain or guttation droplets from hydathodes and stomata, as well as through the
bases of trichomes, cracks in the epidermis and also directly through the leaf cuticle. The areas along the
veins leak more nutrients than other parts of the leaf surface and this is where microbial colonies are often
concentrated.

12. 5. Antagonists and competitors
Among the total micro flora on leaf surfaces, microorganisms compete for nutrients or space or
antagonize each other by production of antibiotics or by mycoparasitism or (exo)lysis of other
microorganisms, or they may stimulate the leaf to produce phytoalexins.
6. Adhesion to leaves
Phylloplane microorganisms have developed different mechanisms for adhering to leaf surfaces exposed
to windy conditions and rain. Yeasts, for example A. pullulans, and other species produce extracellular,
sticky, polysaccharide slimes that prevent their spores and cells from being washed off the leaves.
7. Sources of phylloplane inoculum
The main sources of primary inoculum of epiphytic bacteria, yeasts and filamentous fungi of the
phylloplane of deciduous trees are the overwintering colonies in buds and on twigs, while later in the
season airborne spores settle on the leaf surfaces. Other sources include seed, soil, orchard undergrowth,
air and shoots.· Once yeasts become established, their spores are transferred mainly by water splash and
deposited from air, or by insect, bird and animal vectors.
8. Succession of establishment
The seasonal succession of microorganisms that colonize living leaves. In early spring, the levels of
epiphytic nutrients and airborne inoculum are usually low, which allows the epiphytic bacteria to
predominate in the phylloplane because bacteria take up scarce nutrients more readily than fungal spores
in environments low in nutrients. They can even compete for the nutrient reserves present in fungal
spores, which partially explains the very slow start observed for some fungal spores.

13. Case Studies -1

14. Materials and Methods
1. Isolation and screening of bacteria
2. Dual assay of phylloplane bacteria against papaya fungal pathogens
3. Morphological characterization of the isolates
4. Biochemical characterization of the isolates
5. Evaluation of Individual and Consortium biocontrol potential
6. Preliminary bioassay to evaluate disease control ability of the isolates

15. Result
Bacterial isolates from the phylloplane samples screened for dual plate assay and three isolates
namely IS1, IS6 and IS7 exhibited good percentage of inhibition against fungal pathogen.
IS6 was identified as Bacillus and IS7 was identified as Pseudomonas.
Fruits co-inoculated with IS – 7 and the pathogens showed the maximum freshness. This shows the
significant biocontrol ability of post-harvest diseases of the phylloplane bacterial isolates.
Control (papaya fruits without any treatment) Papaya fruits inoculated with Rhizopus

16. Papaya fruits inoculated with Rhizopus and Isolate 6
Papaya fruits inoculated with Rhizopus and Isolate 7
Control (papaya fruits with any treatment)
Papaya fruits inoculated with Colletotrichum

17. Papaya fruits inoculated with Colletotrichum
and Isolate 6
Papayas fruits inoculated with Colletotrichum and
Isolate 7
Control (papaya fruits with any treatment)
Papaya fruits inoculated with Fusarium

18. Papaya fruits inoculated with Fusarium and
Isolate 6
Papaya fruits inoculated with Fusarium and
Isolate 7

19. Case Studies -2

20. Material Methods
1. DSF* bioreporters construction
2. Isolation and identification of DSF inhibitory bacteria
3. Virulence test
4. Kinetics of DSF degradation in vitro assay
5. Biofilm formation and attachment assay
*Diffusible signal factor (DSF)s

21. Result
To assess the presence and functionality of DSF family molecules in Xcc, two DSF bioreporters were
constructed by transformation of wildtype Xcc 306 and a rpfF mutant with plasmid pKLN55.
The DrpfF strain had impaired DSF production (as expected) and colonies grown on NBY or NA
medium displayed changes in shape (circular to irregular), surface texture (smooth to rough), reduced
mucoidy, and a loss of pigmentation.
A total number of 114 bacterial isolates were isolated from field grown citrus plants with and without
any symptoms of citrus canker, all these isolates screened for their potency to disrupt DSF mediated
induction of gfp expression in the wildtype bioreporter strain Xcc 306/pKLN55. Out of these, total 7
isolates inhibitory effect against DSF signalling pathway and all these classified using API kits and by
sequencing of PCR-mediated 16S rRNA amplification products.
They included two Gram-positive bacteria (Bacillus sp. SJ13 and Bacillus sp. SJ15) and five Gram-
negative bacteria (Pseudomonas sp. SJ02, Pseudomonas sp. SJ01, Raoultella sp. SJ08, Kosakonia sp.
SJ23 and Citrobacter sp. SJ11).
The strains that showed high ability to degrade the DSF signal in vitro were: Pseudomonas sp. SJ01,
Pseudomonas sp. SJ02 and Bacillus sp. SJ13. Its maximum activity was recorded 6 h after DSF
addition. The rate of degradation reached by Pseudomonas sp. SJ01 was 1.3- and 2.2-fold faster than
that reached by Pseudomonas sp. SJ02 and Bacillus sp. SJ15 respectively.

22. Effects of DSF-degrading strains on Xcc virulence
Virulence assays were performed under controlled growth conditions and canker lesions were
quantified at 21 DPI. These assays showed that when citrus leaves were inoculated with mixtures of
Xcc and different DSF inhibitory bacteria by spraying, the number of canker lesions decreased
significantly for three bacteria, Pseudomonas sp. SJ02, Pseudomonas sp. SJ01 and Bacillus sp. SJ13,
and increased for one, Bacillus sp. SJ15, relative to the control inoculated with Xcc alone. When
effects were assayed by leaf infiltration of bacteria, Pseudomonas sp. SJ02 and Bacillus sp. SJ13 still
conferred a significant (at least 2.5-fold) reduction in the number of canker lesions, while Bacillus
sp. SJ15 showed a slight decrease.
Effects of DSF-degrading strains on Xcc attachment and biofilm formation
For most pathogenic bacteria, surface attachment and subsequent biofilm formation are essential
stages in maintenance, survival and early establishment of pathogenicity in tissue.
The results showed that Pseudomonas sp. SJ02, Pseudomonas sp. SJ01 and Bacillus sp. SJ13 strains
significantly reduced the attachment ability of Xcc 306 to abiotic and biotic surfaces, with 8-fold
lower levels of crystal violet stain retention.

23. Figure: Reduction in the severity of citrus canker
disease by the action of inhibitory quorum sensing
bacteria isolated from citrus leaves. (a) Spray
inoculation on the abaxial side of citrus leaves, both
bacteria were co-inoculated at a concentration of 107
CFU/mL
Inoculated leaves were photographed at 21 days
post-inoculation. (b) Inoculation by infiltration.
Right side of leaf: Xanthomonas citri subsp. citri at a
concentration of 104 CFU mL 1, left side of leaf:
DSF inhibitory bacteria isolated plus X. citri subsp.
citri. Both bacteria were co-infiltrated at the same
concentration of 104 CFU mL
The bacterial strains were mixed just prior to
infection. The assays were repeated three times with
three plants each time, yielding similar results. Only
one representative result is presented in the figure.

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