Paper Presented at The International Conference o f Indonesia Forestry Researchers III 3,d INAFOR 2015

Bogor, 21 -22 October 2015___________________________________________________

P A P E R A 2 - E ffe c t F ilte r C o v e r o f S e e d lin g s in D ire c t

U ro m ycla d iu m tep p eria n u m f o r
I n o c u la tio n S c re e n in g o f
F alcataria m o lu cca n a D i s e a s e T o l e r a n t

Asri Insiana Putri1, Liliana Baskorowati1,

Xurhidayati1, Toni Herawan1, Sift I Iusna Nurrohmah1

'Center o f Forest Biotechnolog>- and Tree Improvement Research, JL Palagan Ten tara Pelajar Km. 15,
Purwobinangun, Pakem, Sleman,Yogyakarta, Indonesia, 55582,
Corresponding E-Mail: asriip@yahoo.co.id

ABSTRACT

Effectively o f direct inoculation screening o f F. moluccana to gall rust disease tolerance in

nursery needs to be proved in un-cultured pathogen like U. tepperianum. This technique
requires accurate simulation o f natural environmental conditions for disease infection to host
target TTie aim o f this study is to observed effect o f filter clear poly bag cover in direct
inoculation screening ot F. moluccana by tresh U. tepperianum spores as inoculums. Three
months old o f F moluccana seedlings were used as host target. This research was designed as
four groups o f cases, seedlings with cover and non-cover, gall rust from 400 m asl and from
800 m asl altitudes sites. Each group comprises three families with 10 individual seedlings as
replication. There were significant differences in percentage o f galled formation at all
treatments among family 1 and total families, hut n o significant effect at family 2 and 3 on
NC400 and NC800 treatments. The highest seedlings percentage o f gall rust formation
(94.2480 % ± 1.97468) was in Family 2 with source o f inoculums from 400 m asl. U.
tepperianum spores from lower altitude site source can more able to tormed gall rust than the
higher altitude site.

Keywords: F. moluccana, U. tepperianum, gall rust, filter paper, direct inoculation, screening

1. IN T R O D U C T IO N

Use o f disease tolerant/resistant plants is the ideal m ethod to manage plant diseases, if plants
o f satisfactory quality and adapted to the growing region with adequate levels o f durable
tolerance are available. T he use o f disease tolerance plants eliminates the need for additional
efforts to reduce disease losses unless other diseases are additionally present. Tolerant plants
.ire usually derived by standard breeding procedures o f screening (Fry, 1982; Amcson, 2001;
Maloy, 2005).

O n a global scale, some o f the m ost serious fungal plant pathogens are obligate biotrophic
parasites (Brown Sc Hovmoller 2002). Obligate biotrophic U. tepperianum - F moluccana
interactions were the main obstacle o f gall rust disease tolerant screening. T he term obligate
biotroph characterizes a specific lifestyle in which the host suffers only m inor damage. The
pathogen in turn is dependent on the living host plant to complete its life cycle (Staples 2000).
U. tepperianum produces tcliospores which have ridged longitudinal striations, with three spores
on each head. The size o f the tcliospores ranged from 13-18 gm wide and 17-26 gm long. The
teliospores cannot themselves intect the host; they have to germinate to produce
basidiosporcs, which are formed at least 10 hours after inoculation. Then a penetration peg
was formed by a matured basidiospore 16 hours after inoculation, penetrates the host cells
directly through the epidennis. Seven days after inoculation, vegetative mycelia ot this gall­
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Paper Presented at The International Conference o f Indonesia Forestry Researchers III 3,d INAFOR 2015
Bogor, 21 -22 October 2015___________________________________________________

forming rust give rise to pycnia, recognized as small brown pustule which breaks through the
epidermis. The typical symptom o f gall rust disease on the seedlings is the bending o t the stem
o r shoot, either with o r w ithout the formation o f a dark red necrotic lesion (Rahayu, 2007).

Symptoms o f the disease begins with local swelling (tumefaksi) in the affected part o f the plant
(leaves, branches and stems), further swelling turned into lumps which then became a small
pimple or called gall. Arising galls have varied from round to form irregular diameters ranging
from a few millimeters to greater than 10 cm. I h e galls can be grouped o r spread on the
affected areas. Young gall green light brownish covered by a layer ot powdery slightly reddish
color which is a collection of spores. Old gall reddish brown to black, usually the gall is
porous o r perforated, and is used as a nest o f ants and other insects. When the diseased
section o f the petiole compound o r canopy, that part a little bent because o f thickening and
swelling, then roll up the canopy leaves change shape (malformations) no longer leaves. If the
plants have severe attacks, then the whole plant is filled by the gall, then the leaves dry up
experiencing hair loss, followed by die trunk and branches o f trees and plants eventually die
(Anggraeni, 2008).

The germplasm screening methods can be classified into (i) direct intact o r live plants, (ii)
direct detached plant organs, and (lii) indirect approaches (Steadman et a l, 1997; Olivier et a i,
2008). Excised common inoculation o f F. molttccana has been performed on direct inoculation
using fresh gall rust spores suspensions (Morris, 1987; Kull et uL, 2003). Effective screening
for disease resistance requires accurate simulation o f natural environmental conditions where
plants are exposed to die inoculums (Porta-Puglia & Aragona, 1997). Optim um inoculation
and incubation conditions should be established so that susceptible and resistant genotypes
can be easily differentiated (Infantino et a l, 2006).

Limited availability o f space is often the major constraint to screening in environment control.
Disease evaluation in controlled conditions is often used to identity resistant breeding material
during non-crop periods, but may also be used to confirm the reaction o f tolerant/resistant
genotypes identified in die field or for characterization o f pathogen variability (Infantino et al
2006). Screening in the greenhouse or nursery w ithout plastic cover allows challenging
inoculums in interaction with other phytopathogenic organisms. Even though a major
problem using plastic covers are high moisture, low respiration and the opportunity for cross­
contamination via the w et surface (Xarciso, 2008).

Filter papers were used tor numerous laboratory applications filtration and exhibit such large
proportions o f bacteria and spores in air flow or liquid. Filter paper was first used as a
scientific tool in 1815 by the Swedish chemist Jons Berzelius. O ver the last 50 years, filter
paper has gained an increasingly important role as a substrate for the diagnosis and
surveillance o f infectious diseases (Smir et aL> 2014). Efficiencies for air filtration are normally
expressed as percent penetration or retention for a stated airborne particle size. Particle size
for bacteria is between 0.22 um —30 urn and 3.3 pm for spores (General Electric Company,
2013). Filter paper on plant screening plastic cover can establish natural environmental
conditions lor disease target development like humidity, respiration and temperature, without
interaction with other phytopathogenic organisms.

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Paper Presented at The International Conference o f Indonesia Forestry Researchers III 3,d INAFOR 2015
Bogor, 21 -22 October 2015___________________________________________________

2. EX PERIM ENTAL M E T H O D

2.1 Seedlings an d gall m st spores m aterial

Sengon seeds were collected from Wamena, Papua. Seedlings direct inoculations were
observed in tissue culture nursery, Center o t Forest Biotechnology and 1'ree Improvement at
Kaliurang, Yogyakarta (7°35’50.44” SL and 110°25’33.54” EL), 800 m asl. altitude, 21 °C daily
average temperature, 85 % daily humidity, 28-9200 lux daily light and daily win speed 0-0.4
m /s. Gall rust as U. topperianum as spores source collected from 2 site o f sengon society forest,
Kaliurang, Yogyakarta (7U35’50.44” SL and 110°25,35.54” EL), 800 m asl, altitude and Tridadi,
Yogyakarta f7°4T83.28” SL and 110°21 *97.82” EL) 800 m asl. Research activities were
observed from 2012 until 2014.

2.2 Filtered nursery d irect inoculation

Direct inoculation in this research was based on Morris (1987) and Kull d a l. (2003) method
with some modifications. Fresh spores were inoculated directly into all parts o f healthy 3
months old seedling, two rimes for a week. Concentration o f spores predetermined by
haemocytometer at least lxK P spores/m l in sterilized water containing 0.2 ml/1 Tween"0
(polysorbate surfactant), stability and relative non-toxicity emulsifier. A fter inoculation, the
seedlings covered by perforated clear poly bag with Watman2" filter paper for 14 days. During
incubation, seedlings moisture was maintained by capillarity system with water in the beds.
Pest prevention was maintained by spraying insecticides in nursery every 1 week (Figure 1).

O p e n th e cover
a f t e r 14 d a v s

D ire c t in o c u latio n

f t
I g a ll ru s t

/ / . te p p e r in n u m S e e d lin g m e d ia in
s p o re s s o lu tio n b la c k p olv b a g
V
Figure 1: Direct inoculation in nursery o f obligate biothropic pathogen

2.3 D esign experim ent

'Ibis research was designed as four groups o f cases, seedlings with cover and seedlings with no
cover, gall rust from 400 m asl and from 800 m asl altitudes sites. Each group comprises o f
three families with 10 seedlings as replication. Parameter o f this observation was percentage o f
seedlings that attacked by gall rust. All treatments were compared with control. Ihe group
means data were analyzed using post hoc tests in the Analysis o f Variance (ANOVA), if
significant differences existed between the treatments, then groups will be analyzed using
Duncan’s multiple range tests.

3. RESULT A N D DISCUSSION

Direct inoculation technique for each o f the two sources o f U. tepperianum spores was used to
infected /'. moluccana seedlings individually. Figure 2 illustrated calculated and inoculated the

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Paper Presented at The International Conference o f Indonesia Forestry Researchers III 3,d INAFOR 2015
Bogor, 21 -22 October 2015___________________________________________________

fresh spores with filter poly hag cover and one month after the cover is opened. This directs
intact inoculation application was modification technique lo r L'. teppmamtm live plants in
nursery for screening disease tolerant. Successtul screening for disease tolerant is based on the
availability o f precise and accurate screening technique (Infantino et a l, 2006).

Figure 2: Gall rust o f sengon with red-brown spores (a), fresh spores sampel in
sterilized w ater (b), spore calculation with haemacytometer (c), sterilized w ater containing 0.2
m l/l Tween20 and at least 1x10s spores/m l (d), microscopic observation o f U. teppmamtm
spores (e), seedling media polybag covered by perforated plastic bag with Watman20 filter
paper (f), seedlings inoculation and covered by filtered perforated plastic (g), sengon seedlings
after one month inoculation (h).

Table 1: Descriptive statistic o f each sengon family separately and the treatments

T r e a tm e n ts (% ) ± S E ‘
F a c to r
C 400 C 800 N C 400 N C 800

Family 1 86.9590 ± 3.79931 52.0590 ± 2.23692 20.0250 ± 1.22679 10.1620 ± 0.42476

Family 2 94.2480 ± 1.97468 54.2720 ± 3.02714 15.9250 ± 1.78243 10.4530 ± 0.40555
Family 3 89.1140 ±3.43941 54.3730 ± 2.66821 16.2350 ± 1.61897 10.4270 ± 0.42460

S ta n d a t'd erro r o f th e m ean

The descriptive statistic for each family separately and the overall treatments were shown in
Table 1. TTie result showed the highest percentage o f seedlings that attacked by gall rust was in
(MOO, followed by C800, XC400 and the lowest was XC800 treatments for all tamilies. It is
often difficult to achieve high and uniform pathogen pressure in the field year after year to
permit adequate evaluation o f plant gcrmplasm for disease avoidance or physiological
resistance because both are confounded under field conditions (Miklas & Grafton, 1992). The
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Paper Presented at The International Conference o f Indonesia Forestry Researchers III 3,d INAFOR 2015
Bogor, 21 -22 October 2015___________________________________________________

m icro en v iro n m en ts like filter c o v e r a t nursery in this research can be expected to reduce the
difficulty o f p lan ts disease screen in g to discrim inate betw een physiological resistance and plant
architectural av o id an ce o t pathogen.

T a b le 2: R esum e o f A N O V A te s t resu lts fo r p ercentage o f seedlings th a t attack ed w ith gall

ru st disease

Source o f Variation Degree of Freedom Sum of Squares Mean of Square F Sig.

Family 1 Between 3 36186.910 12062.303 228.409 0.001”
Groups
Within 36 1901.165 52.810
Groups
Total 39 38088.075
Family 2 Between 3 45436.787 15145.5% 369.303 0.001"
Groups
Within 36 1476.405 41.011
Groups
Total 39 46913.192
Family 3 Between 3 40323.551 13441.184 247.192 0.001"
Si

36 1957.521 54.376
Groups
Total 39 42281.072
Control 3 0.001 0.001
** Siyiificant at 95% confideut internallevel

'Fable 3: R esum e o f D u n c a n ’s M ultiple Range ’le s t results fo r percentage o f seedlings th a t

attack ed w ith gall ru st disease.

Subset for alpha 0.05

Treatments
a b c d
Family 1
C400 86.9590
C800 52.0590
NC400 20.0250
NC800 10.1620
Family 2
C400 94.2480
C800 54.2720
NC400 15.9250
NC800 10.4530
Family 3
C400 89.1140
C800 54.3730
NC400 16.2350
NC800 10.4270
Total Family
C400 86.9590
am 52.0590
NC400 20.0250
NC800 10.1620
Control 0.000 0.000 0.000 0.000
Remarks: Numbers followed by different letters, a, b. c or d in the same column indicate significant differences
(P<0.05) according to Duncan's Multiple Range Test

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Paper Presented at The International Conference o f Indonesia Forestry Researchers III 3,d INAFOR 2015
Bogor, 21 -22 October 2015___________________________________________________

ANOVA test, determine the effect o f treatments to the percentage o f seedlings that attacked
with gall rust disease (I'able 2), indicated the significant ettect o t filter cover, the inoculums
sources and the combination o f these treatments individually between sengon families.

More specific from the further test using Duncan’s Multiple Range Test (Table 3), it was
shown that all treatments at family 1 and total family had significant effect but at family 2 and
3 had no significant effect on XC400 and XC800 treatments. This result indicated that die site
o f inoculums source have a role o f pathogen infection and gall rust formation. T he severity o f
this disease depends on the interaction o f the gall rust tungus, host response, and
environmental conditions. T he disease developed rapidly is probably due to favorable
environmental conditions (Franje e ta l, 1993; Braza, 1997; Old & Cristovao, 2003).

Famili I
ITU -
*

U m ,i||

i i : J III llljim linrftTt-._

44H> ('800 NC400 NC800 C
* „ c
Treatments

Famili 2

*
3
Jl — inn it

M H r u

r Cfl
C4oo cm NC400 mm c

T r e a tm e n ts

a b

Figure 3: Percentage o f seedlings in gall rust attacked from family 1 (a), from family 2 (b),
from family 3 (c), stem o f sengon seedling attacked by gall rust (d) and petiole o f sengon
seedling attacked by gall rust (c)

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Paper Presented at The International Conference o f Indonesia Forestry Researchers III 3,d INAFOR 2015
Bogor, 21 -22 October 2015___________________________________________________

Fresh spores o f un-cultured biotrophic pathogen from nature have a limited life time outside
the host, thus the study ot this technique is important to obtain accuracy o f infection in fast
incubation. This direct inoculation technique indicated that filtration through common filter
paper (Whatman 40) on clear poly bag cover o f seedlings have higher percentage o f gall rust
attacked. The inoculations technique led to gall rust formation during 9 months incubation for
all families and treatments but there was no attacked at all in control. Figure 3 shown that
percentage o f seedlings in gall rust attacked has similar trend line in sengon family 1, sengon
family 2 and sengon family 3. These similarity shows that the technique can be repeated in
more than one population. Repetition is the requirements to r the validity ot new technique or
modification technique. However, the technique must be observed for other un-cultured
biotrophic pathogen in interaction with specific h o st plant.

Plants have the ability to continuously respond to various stimuli which alter their physiology,
morphology and development. These range stimuli are from essential to toxic in their effects
(Tugizimana et al, 2012). A clear and broader understanding o f F. moluccana tolerant for gall
rust screening that caused by U. tepperiannm has not been fully investigated in all aspects, for it
would open up possibilities o f developing novel, more effective and sustainable strategies to
control or eradicate fungil diseases in plants.

4. C O NCLUSIO N

Filter cover o f seedlings in nursery direct inoculation were effective as alternative technique
for Falcatana moluccana gill rust tolerant screening that caused by Uromycladium tepperiannm, the
obligate biotrophic parasites pathogen.

ACKNOWLEDGMENT

The author would like to thank Fithry Ardhani, l itis Budi Fkowati as researcher team for
helpful discussions. We are grateful to Endin Izudin, Suprihati, Rudi H artono, Waluyo, Alin,
Margiyanti, Heri, Sukijan and Susanto for technical assistants.

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