Viral Splash

Viral Splash (or so-called color break symptom) may be found in any plants which are infected with Frangipani
Mosaic Virus (FrMV).   FrMV replication and activity in plant causes a change in color pattern of pigmented flower
and leaf by overaccumulation of epidermal anthocyanin pigments.  In this case, anthocyanin may play role in plant
defense mechanism against pathogen (i.e. FrMV) which is active in that particular area.

In general, viral splashes in most plumeria flowers can be distinguished from other kinds of splashes (1) by their
intense discolored areas (due to the accumulation of anthocyanin in the area of high viral activity) on the flower
petals i.e. dark red/maroon splash on red flower, darker pink on pink and light pink flower, red on yellow flower,
and dark purple on purple flower, (2) by their uneven/unpredictable forms and sizes (vary widely in shape and
size), (3) by their unpredictable locations, and (4) by its non-consistant patterns (non-repeated patterns) from
petal to petal and flower to flower (Fig. 13 and 14).   On foliate, the virus also unevenly causes the deficiencies in
chlorophyll pigments resulting in mosaic appearance (non-uniform chlorosis).  The viral symptoms on leaf may
also include stains, discoloration and mottled appearance due to anthocyanin accumulation (Fig. 15 and 16). One
important feature of the viral mottled makings on leaves is that their appearance (color and pattern) keeps
changing as the leaves expand and as the symptom progresses into other stages.  The color of viral symptoms
generally fades when the leaves age.  The severity of FrMV infection and its symptom on flowers or leaves can
vary greatly depending on the strain of FrMV, the cultivar/specie of the plumeria (host plant) as well as the
environmental conditions such as temperature.

If Miracle plumeria (having natural splash) and Fantasia or Maya (having chimeral splash) plumerias are infected
with FrMV, the viral splash (symptom) may also appear on these plants and this can complicate the interpretation
of the splashes (Fig. 17-21).  Splash which is located beyond the boundery of natural splash is likely due to FrMV
or other means such as insects, fungus, chemicals and etc.  The anthocyanescent splashes or markings
associated with the infections of insects and etc. are unique and differ from the splash caused by the FrMV.

As FrMV is easily transmitted by physical contact (via sap), it can spread rapidly and covertly in plumerias with
poor cultural and sanitation practices (such as using unsterilized/contaminated cutting tools between plants or
grafts).  It may lie latent for many months or longer before expressing its symptom.  Viral infections of plants are
incurable.  Any plant that is vegetatively propagated from a virus-infected mother plant will also be infected.  This
virus now has a worldwide distribution via infected cuttings, grafts and plants which have caused once-rare
disease in nature to become a more common disease in cultivation.

High temperature can put a lot of stress on the plumerias as well as provides an ideal condition for FrMV to be
more active, and flavours FrMV's replication within a plant.  FrMV is the most active at 30-35C or 86-95F.   It is
important to note that heat does not cause this splash to occur (only provide a favorable condition) but the virus
present in the plant does.  For more information on FrMV, Click
FrMV in plumeria
Variegation in plants is described as the appearance of differently colored zones in the
plant's organs such as leaves or flowers, and it is mostly manifested as stripes, blotches,
steaks or splashes.  The word "Splash" has been widely used to describe pigmentation
patternings or variegations in plumerias by a number of plumeria glowers.  Splash such as
natural splash is a form of natural colorations which is genetically regulated but its phenotype
can be affected by a number of factors, and is often mistaken for other kinds of splashes or
vice versa.  Differentiating between virus induced symptom (viral splash), natural coloration
(natural splash) and variegated mosaics due to shoot apical meristem layer-specific
mutations (chimeral splash) in plumeria can be challenging.  This requires experience, and
many times it needs to be investigated scientifically in laboratory.  Below is a brief overview
on basic characteristics of these splashes and a few similar variegations which are found in
plumerias, and how they arise.
Chompoo Paan plumeria
Kukiat's Garden
Splashes in Plumeria
Natural Splash

Natural colorations occur in a plant that consist of cells having identical genotype but only the genes in specific
plant's parts (e.g. leaf, flower, stem, root or the specific cell layers and etc.) are instructed to express or not to
express the colors (or to make or destroy the color pigments).   Due to the differential gene expression, plant with
the homogeneous genotype can have different color for each organ (e.g. leaf can be green while flower can be red,
yellow or white and etc.).  At the petal level, the petal can have different color in a different location.  This kind of
differential gene expression is the most common cause of floral coloration patterns in plants including plumeria.

Of all the natural colorations found in plumeria flowers, a highly unstable form of natural colorations is also present
on specific regions of petals in some plumeria cultivars.  The term "Natural Splash" is applied here to describe an
unstable phenotype of flower coloration which is characterised by unstable expression of a gene located at specific
location of flower petals.  The gene expression which is highly unstable can be influenced by a number of factors
such as location (position) on plant's parts (e.g. specific area on petal) and stages of plant development (e.g.
flower's age and incurvation of petals) and etc.   Whereas plant's regulatory gene determines the coloration of
natural splash in flowers, one (or more) of a variety of factors such as the environmental conditions (e.g. plant
stress, temperature, light intensity) and cultivation practices (e.g. plant nutrition) affect the plant development and
many aspects of plant physiology, which in turn has an impact on gene expression in some extend (i.e. more or
less pronounced splash or in some cases with no splash at all).

Plant molecular geneticists relate this phenomenon under certain condition(s) to the switching (partly or totally) "on
and off" of color gene in that particular location of the plant's parts (e.g. specific area of petals).  The other words,
the switches govern gene expression by controlling where, when, and even to what degree a particular gene is
turned on.  Since natural splash is
genetically regulated and geographically predetermined, it has a predictable
coloration pattern and occurs at about the same location on each petal.  In addition, its coloration and splash
pattern at a given condition are consistant (repeated pattern) on every flower (note: not appearing randomly like a
virused plant).

Splash in cultivars such as Miracle, Madame Poni, Madame Rainbow, Buddha Raksa, Gam Maam and Kasetsin
plumerias fall into this category i.e. red stripe/splash over a part of off-white or yellow area of petals.  This is the
only area on petals where the genes are instructed to have the natural red splash (relevant mainly to anthocyanin
biosynthesis) (Fig. 1-4).  Since stages of plant development (e.g. incurved petal), environmental factors and etc.
can alter how the genes in this particular location behave, the coloration of the splash under these conditions may
vary i.e. lighter or darker in color, and more or less area of splash or no splash at all.  In this case, natural splashes
on these flowers express as a part of floral coloration patterns.  A good example of natural splash in other plant is
the stripes on watermelon rinds.
Chimeral Splash

Unlike natural splash, chimeral Splash (or so-called variegation) occurs in plants with two or more genetically
distinct types of cells (i.e. mutated and non-mutated cells) that coexist in a plant but express differently.  It can
involve gains or losses of color pigment(s) in the mutated cells.  In case of plumeria, mutated cells are generally
associated with the absence of pigmentation in flower and leaf.  This is mainly due to the inhibition of anthocyanin
or chlorophyll biosynthesis.

Somewhere early in the development of a flower and leaf, any genetic mutation in cell(s) in a shoot apical meristem
(which gives rise to plant body) can result in the generation of chimera.  The location and pattern of chimeral splash
on a petal and leaf directly relate to where in the meristem, the mutated cells are located; and how these cells
duplicate themselves (cell division) during plant's growth.  Due to the differing planes, durations and rates of cell
division amongst mutated and non-mutated cells while the flower and leaf being formed, chimeral splash is irregular
in coloration pattern and shape, and appears at unpredictable locations on plant's parts.  The most commom feature
of chimeral splashes on flowers is the narrow white colored stripes running along the petal length (Fig. 6, 7 and 8).

In case of a flower, splash in cultivars such as Fantasia and Marble Ice Cream plumerias fall into this category i.e.
white (colorless) stripes on the petals are due to the presence of mutated cells in white/colorless area whereas a
sort of washed-out pink/red colored stripes are due to the arrangement of both mutated and non-mutated cells in
such areas (Fig. 6).  In case of foliage, Maya and Marble plumerias are examples that fall into this category (i.e.
variegation seen on leaves is due to the alignment of mutated and non-mutated cells in meristematic layers that give
rise to the leaf tissue) (Fig. 10).  These chimera plumerias are rare in occurrence and cannot be seed propagated.

The variegated-leaved plumerias from Thailand that are available in the market are chimeral plants.  The periclinal
chimera (which is relatively stable and can be vegetatively propagated as the whole plant is entirely chimeric) is
discovered by removing an axillary bud (having mutated cells at the growing point) from a branch (with mericlinal or
sectorial chimeras) and grafting it to a root stock (using bud grafting technique).   After sorting and repeating the
process over and over for a period of time, Thai growers successfully created periclinal chimeral plumerias with
unique foliar patterns of variegation (Fig. 12).

Figures 5 to 12 illustrate the various phenotypes (observable characteristics) of chimeral splashes found in flowers
or leaves of plumerias.  It is important to note that some phenotypes may go unnoticed as chimera cells can be
located in deeper layers and masked by overlaying tissues.  With the diversity and complexity in nature of chimeras,
it can be misinterpreted if it is done without scientific investigation in the laboratory.  Since there are very limited
information related to chimera in plumeria, more studies especially at the molecular level should be conducted to
provide a more accurate classification of these chimeras.
Figure 6. Floral chimera splashes in plumerias - Left (Fantasia plumeria) and Middle (Marble Ice Cream plumeria):
These are possibly non-patterned sectorial chimera that mutated cells are scattered randomly throughout the layers of
both apical and inflorescence meristems that give rise to petals.  White colored stripes on their petals are the area that
the mutated cells are present whereas the area with mixed colors (white and light pink/red) are due to the arrangement
of mutated and non-mutated cells.  Although these chimeral stripes are non-repeated patterns, the plumerias with this
kind of chimera can be vegetatively propagated (i.e. from stem cuttings).
Right: Fringed petals with white chimeral
stripes in Hana Emi plumeria.  The irregular edges of petals at which the white stripes are present are the result of the
differences in cell division rate and duration between chimera cells (white region) and normal cells (pink region) during
petal development.  Similar irregular petal edges are also found in Fantasia and Marble Ice Cream plumerias.
Figure 10. Periclinal chimera in plumerias - exhibiting uniform and stable variegation which can be vegetatively
propagated (e.g. from cutting) as the whole plant is entirely chimeric.
Left: Marble plumeria, Middle: An unknown
variegated plumeria,
Right: Maya plumeria
Figure 3. Natural splash in Purple Serendipity plumeria governed by unstable color expression gene - Left: No
natural splash,
Middle: With natural splash in summer season (green arrow), Right: The natural splashes (if any)
that appear on petals are always at the same location in all flowers and have consistant pattern from flower to flower
(repeated pattern).
Figure 17. Natural splash vs viral splash on the same flowers of Miracle plumeria - Left: Miracle with only natural
splash,
Middle and Right: Viral and Natural splashes on the same flowers of Miracle plumeria at the same time.
Viral splash is distinguished by its dark red/marroon in color -- indicated by the arrows.  Natural splash is the lighter
red bands that appears on one side of petals in all flowers and has consistant pattern from flower to flower (repeated
pattern).  Whereas viral splash displays a ramdom color splashing with uncharacterized forms and sizes and at
unpredictable location, and its pattern is non-consistant from petal to petal and flower to flower (non-repeated pattern).
Figure 18. Natural splash vs viral splash on Madame Poni plumeria - Left: Madame poni with natural splash (a
narrow stripe (repeated pattern) running almost the length of each petal).  
Middle: Viral splash (black arrows) and
natural splash (green arrows) on the same flower,
Right: In this case, natural splash (if any) is likely musked by viral
splash which is characterized by its highly unpredictable forms and sizes and at unpredictable location, and its
pattern is non-consistant from petal to petal (non-repeated pattern).
Figure 1. Natural splash in Madame Poni plumeria governed by unstable color expression gene - Upper left:
Intense reddish splash only within incurved area,
Upper middle: Less pronounced red splash to almost no splash
in incruved area,  
Upper right: Less pronounced splash in non-incurved flower, Lower left (Flowers with incurved
petals) and
Lower right (Flowers with non-incurved petals):  As shown in these pictures, the natural splashes (if
any) that appear on each petal are always at the same location and their pattern is consistant from flower to flower
(repeated pattern).
Figure 2. Natural splash in Miracle plumeria governed by unstable color expression gene - Left: Intense reddish
natural splash located on one side of each petal,
Middle: Less pronounced red splash on one side of each petal,
Right: The natural splashes that appear on petals are always at the same location in all flowers and have consistant
pattern from flower to flower (repeated pattern).
Figure 9. Mericlinal chimera in plumerias - only some parts of plant are chimeric. Left, Middle and Right:
Variegated and normal (green) branches in the same plant.  This type of chimera tend to be unstable and in time
one genotype will out compete the other causing the tree to revert either completely green (normal plant) or other
types of chimera plant.
Figure 12.  Creating stable periclinal chimeras - Left image: Plumeria stems with sectorial chimera that is highly
unstable.  Mutated cells extend vertically through all cell layers within the bud or meristem. The border between
each genotype, i.e. nonmutated cells (normal green) and mutated cells (no chlorophyll pigment), is almost parallel
within the stem giving sectorial chimeras a candy cane like appearance.  By removing and grafting an axillary bud
that develops in the axil of a variegated leaf (as seen in the
Middle image) to a rootstock, one may get a periclinal
climeral plumeria with unique foliar pattern of variegation (plant on the left of the
Right image) which is relatively
stable and can be vegetatively propagated.
Figure 5. Sectorial floral chimera splashes in plumerias - Left: One petal of flower (white petal) made up one
different cell type (i.e. mutated cells which stopped producing the usual pink/red pigment).  
Middle and Right:
Sometimes both genotypes (colors) are expressed together in individual petals giving them a beautiful split
coloration pattern.  This type of chimera is very unstable and can not be vegetatively propagated.
Figure 19. Natural Splash vs Viral Splash on Gam Maam plumeria - Left: Natural splash (located at the center of
the flower and is consistant from petal to petal),
Middle: Natural splash (only at the center which is its traditional
location) with viral splash (at unpredictable locations on the petals), and
Right: Viral splashes on yellow colored
petals (in this case there is no natural splash on Gam Maam flower as the unstable expression color gene turns off).
Figure 11. Chimera in plumerias - Left and Right: Possibly non-patterned sectorial chimera with random
colorless patches or stripes
Figure 8. Rare, complex types of floral splashes (variegations) - Left: Twin stripe (white and red regions on a
pink petal) in Charlotte Ebert likely descended from a somatic cell that underwent somatic crossing over
(interchange of chromosomal parts resulting in the change of color expression of this floral mutated gene) during
cell division.  The mutated cells in white area are associated with the absence of pigment while those in red area
are associated with the increase in anthocyanin biosynthesis.  The normal pink area which constitutes
non-cross-over tissue (normal tissue) remains pink in color.  
Right: Mixed types of floral variegations i.e. sectorial
chimera (solid white region on a petal) and other unknown types of variegation (possibly caused by either somatic
crossing over or transposons (jumping genes) resulting in the change of color expression of floral color genes).
Figure 21. Viral symptoms on variegated (chimeral) leaves - Left: An early stage of viral symptom on leaf of a
variegated plumeria,
Right: Stains and discoloration (light brownish red splotchy markings over the entire leaf area) on
leaf of variegated Namwan plumeria.
Figure 14. Viral symptoms on flowers of various plumerias - Left: Dark purple splash on flowers of virused
Muang P-26 plumeria,
Middle: Red splash on flowers of virused Dwarf Singapore Pink plumeria, Right: Red
splash on a flower of virused Bali Whirl plumeria
Figure 13. Viral symptoms on flowers of various plumerias - Left: Red splash on a flower of virused Veracruz
Rose plumeria,
Middle: Maroon splash on flowers of virused J-105 plumeria, Right: Red splash on flower of
virused Vishanu Gold plumeria
Figure 4. Natural splashes in various plumeria cultivars governed by unstable color expression gene - Left:
Natural splash (located at the tips of each petal) in Tempest plumeria,
Middle: Natural splash (a narrow stripe
running almost the length of each petal) in Buddha Raksa plumeria,
Right: Natural splash (located within incurved
area of each petal) in Sangria Stripe plumeria.  As the natural colorations are governed by unstable expression
gene, a number of factors may have an impact on their phenotypes of these splashes under various conditions.  As
shown in these picture, the natural splashes that appear on petals are always at the same location and their pattern
is consistant from flower to flower (repeated pattern).
Viral splash and Natural splash on the same flowers
Viral splash and Chimeral splash on the same flowers or leaves
Figure 20. Viral splash on Chimeral splashed flowers: Under construction
Figure 16. Various viral symptoms on leaves - Left: Stain and discoloration on leaf of virused Dwarf Singapore
Pink plumeria,
Middle: Stains and discoloration on leaf of virused Puddha Raksa plumeria, Right: Reddish
patches/spots on petioles of virused Coral Cream plumeria
Figure 15. Various viral symptoms on leaves - Left: An early stage of viral symptom (mottling) on newly emerging
leaves (which has yet to fully unroll - indicated by the arrow) of Vera Cruz Rose plumeria.  The lesion develops
further into different forms as the viral activity progresses during leaf enlargement and maturation.,
Middle: Severe
leaf malformation and mottled leaves of Leuang Sa-ngeam plumeria.  Notice an early stage of viral lesions
(indicated by the arrow)on a newly emerging leaf which normally go unnoticed,
Right: Mosaic and faint chlorotic
lesions on leaf of Kled Tabtim plumeria
  Natural Splash
Chimeral Splash
Viral Splash
Splashes on Flowers
Splashes on Leaves
 
Genotype
Homogeneous type of cells
(all are normal cells)
Two or more genetically
distinct types of cells
(normal and mutated cells)
N/A

(virus is able to infect plants
regardless of cell types)
A form of anomaly?
No

(It is a highly unstable form of
natural coloration controlled by
unstable expression gene)
Yes

(related to mutation)
No

(It is a symptom of viral infection.)
Location of splash
Always at the same location

(splash (if any) is always at the
same location as it is genetiaclly
regulated and geographically
predetermined)
Unpredictable location

(splash/stripe is only at the area
where the mutated cells located)
Random

(appears randomly on plant's parts
which are the areas where the
virus is active)
Pattern of splash
Repeated pattern
(consistant from petal to patal and
flower to flower)

(The pattern seen is the result of
differential gene expression
between genetically identical
cells)
Unpredictable pattern

(The pattern seen is the result of
the arrangement of two or more
genetically distinct types of cells.

It also depends on the direction of
the growth of mutated cells, and
the difference in rate and
duration of cell division between
mutated and normal cells.)

In flowers (for an example), it is in
the form of stripes
running along
the petal length
(Fig. 6-8).
Non-repeated pattern
(non-consistant pattern)

(The patterns of viral symptom
vary within the same plumeria
cultivar and from cultivar to
cultivar.  Moreover, they are not
consistant from petal to petal and
flower to flower.)
Coloration of splash
(on flower/leaf)
Red splash (on flower)
(due to anthocyanin biosynthesis)
Colorless (in most cases)
(due to the absence of pigments
in mutated cells)

Flowers and Leaves:  The overall
variegation may fade with age but
the chimera pattern remains the
same.
Intense discolored areas on
flowers
(due to anthocyanin accumulation.)

Symptom on flowers: Dark
red/maroon splash on red flower,
darker pink on pink and light pink
flower, red on yellow flower, and
dark purple on purple flower

Stains, discoloration and
mottled appearance on leaves
(due to anthocyanin accumulation
or chlorophyll deficiency.)

*The viral markings on leaves keep
changing in appearance (color and
pattern) as the leaves expand and
as the symptom progresses into
other stages.

**The symptoms are NOT necessary
appear on on flower and leaf at the
same time.
Splash present on
plant
come and go, and reappear
(but always reappear at the same
location on petals)

(As it is govern by the unstable
expression gene and affected by
the environmental conditions)
come and go, and may or may
not reappear

(depend on the location of
mutated cells within plant's
tissues)

(Chimera splash can be seen
when the mutated cells located at
the outside of plant tissue.  But it
cannot be seen when the mutated
cells located deep inside the plant
part.)
come and go, and reappear
(but reappear at random location)

(depend on the activity of virus
and environmental conditions.  
FrMV resides in infected plant
throughout the plant's life;
therefore, the symptom will
reappear when the condition is
right.)
Irregular Petal Edges
(Fringed Petals)
No
(All are identical type of cells that
have the same rate of growth)
Yes
(The growth rate (cell division) of
mutated cells is inferior to that of
normal cells.  As a result, the
irregular (fringed) edges on
flowers' petals are formed.)
No
(it is nothing to do with the rate of
cell division or growth)
Vegetative
Propagation (e.g.
cutting)
Yes
Yes for Periclinal chimera plant
N/A
Cuttings taken from an infected
plant are also infected.
Seed Transmission
Natural splash is a result of gene
expression.  It is variegation
typical of cultivars/species, and it
is a characteristic that a plant
can randomly pass on to its off
springs through its genes.
This type of chimera cannot be
seed propagated.  As chimera
occurs in apical cell layers,
chimera variegation is not
variegation typical of
cultivars/species, and cannot pass
on to its off spring.
FrMV does not transmit through
seed, resulting in absence of virus
in seedlings.
Infectability
No
No
Yes
(by mechanical means via sap)
Occurance
Rare
(seen in some cultivars)
very Rare
Rare in Nature but commonly
seen in cultivation


(Spread has occurred through the
use of virus-infected cuttings
during propagation.  The
distribution of virus-infected
material (cuttings and grafts) in
international trade has made
matters worst.)
       
Table 1. Comparison of some key characteristics of Natural, Chimeral and Viral Splashes
Figure 7. Rare green chimeral stripes on yellow petal background in Shirakawa Yutaka's plumeria seedlings.  In this
case, the mutated cells in the green area are associated with the increase in chlorophyll biosynthesis  The differences
in the rate and duration of cell division between chimera cells (green region) and normal cells (yellow region) during
petal development resulted in the irregular (fringed) petal edges on these flowers' petals.  The stability of this floral
chimera remains to be seen.