Revista Mexicana de Fitopatología
ISSN: 0185-3309
[email protected]
Sociedad Mexicana de Fitopatología, A.C.
México
Gabino Gutiérrez, Rodrigo; Carrillo Castañeda, Guillermo; Fucikovsky Zak, Leopold
Identification of Bacteria Associated to Necrotic Leaf Spots in Plants of Lycaste aromatica Lindley
Revista Mexicana de Fitopatología, vol. 22, núm. 3, diciembre, 2004, pp. 338-344
Sociedad Mexicana de Fitopatología, A.C.
Texcoco, México
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Identification of Bacteria Associated to Necrotic Leaf Spots in
Plants of Lycaste aromatica Lindley
Rodrigo Gabino-Gutiérrez, Guillermo Carrillo-Castañeda, Colegio de Postgraduados
(CP) Programa de Genética, Instituto de Recursos Genéticos y Productividad, km 36.5
Carr. México-Texcoco, Montecillo, Edo. de México 56230; and Leopold FucikovskyZak, CP, Instituto de Fitosanidad, Programa de Fitopatología. Correspondence to:
[email protected]; [email protected]
(Received: June 28, 2004 Accepted: September 07, 2004)
Gabino-Gutiérrez, R., Carrillo-Castañeda, G., and
Fucikovsky-Zak., L. 2004. Identification of bacteria
associated to necrotic leaf spots in plants of Lycaste aromatica
Lindley. Revista Mexicana de Fitopatología 22:338-344.
Abstract. Plants are colonized by an array of microorganisms
that have deletereus, neutral or beneficial effects on growth
of the host plant. In order to obtain preliminary information
about the leaf-associated bacteria of the orchid Lycaste
aromatica, leaves exhibing foliar necrotic spots from ten
plants were collected in Comitan, Chiapas; Cuetzalan, Puebla,
and Cordoba, Veracruz, Mexico. Forty two isolates were
obtained; their genera and proportion were as follows:
Bacillus, 38% (Cuetzalan 9%, and Cordoba 29%);
Enterobacteriaceae, 36% (Comitan 10%, Cordoba 24%, and
Cuetzalan 7%); Pseudomonas, 7% (Comitan 5%, and
Cordoba 2%); Xanthomonas, 5% (Cordoba); Clostridium,
2% (Cordoba), and bacteria with coccus morphology, 12%
(Cuetzalan 5%, and Cordoba 7%). None of the isolates were
pathogenic and based on the total indol production test, none
were plant growth promoters through auxin production;
however, Bacillus, Enterobacteriaceae, and Pseudomonas are
known for their antagonistic capacities against other
microorganisms.
Additional keywords: Orchid, Bacillus, Pseudomonas,
Enterobacteriaceae, Xanthomonas, Pantoea.
Resumen. Las plantas son colonizadas por una diversidad
de microorganismos que tienen efectos deletéreos, neutrales
o benéficos en el crecimiento de las plantas hospederas. Con
el fin de obtener información preliminar sobre las bacterias
asociadas a las hojas de la orquídea Lycaste aromatica, se
colectaron hojas exhibiendo manchas necróticas en las
cercanías de Comitán, Chiapas; Cuetzalan, Puebla, y Córdoba,
Veracruz, México. Se obtuvieron 42 aislamientos bacterianos
cuyo género y proporción fueron: Bacillus, 38% (Cuetzalan
9% y Córdoba 29%); Enterobacteriaceae, 36% (Comitán
10%, Córdoba 24% y Cuetzalan 7%); Pseudomonas, 7% (5%
Comitán y 2% Córdoba); Xanthomonas, 5% (Córdoba);
Clostridium, 2% (Córdoba), y bacterias con morfología de
coco, 12% (5% Cuetzalan y 7% Córdoba). Ninguno de los
aislamientos resultó patogénico y con base en la prueba de
producción de indoles totales, ninguno es promotor del
crecimiento vegetal mediante la producción de auxinas; sin
embargo, Bacillus, Enterobacteriaceae, y Pseudomonas son
reconocidos por su capacidad antagónica contra otros
microorganismos.
Palabras clave adicionales: Orquídea, Bacillus,
Pseudomonas, Enterobacteriaceae, Xanthomonas, Pantoea.
The genus Lycaste comprises important American orchids,
for example: L. virginalis (Scheidweiler) Linden is the
national flower of Guatemala. Eight species of this genus are
present in Mexico, of which L. virginalis and L. depeii (Lodd)
Lindley have ornamental importance (Pelham, 1958), and L.
aromatica Lindley, one of the species widely distributed in
Mexico, and L. cruenta Lindley are important in the orchid
scent industry (Kaiser, 1993). These species under cultivating
conditions, may present foliar necrosis which spoils the plant
appearance and reduces the photosynthetic area. The necrotic
leaf spots show a yellow halo, which initially developed in
extended form between the main ribbings, which are typical
bacterial disease symptoms (Montesinos, 1996). Plantassociated bacteria cause deleterious effects on host plant
growth. Bacteria also play an important role in the
conformation of the populations of microorganisms
establishing synergistic or antagonistic interactions that limit
or promote their development (Schaad et al., 2001). These
interactions can have diverse beneficial effects for the plant
(Barazani and Friedman, 1999; Barea et al., 2002;
Buchenauer, 1998; Carrillo-Castañeda and Alvarado-Cano,
2000; Carrillo-Castañeda et al., 2003; Dobbelaere et al., 2003;
Gupta et al., 2002; Hammerschmidt et al., 2001; Kurek and
Jaroszuk-Scisel, 2003; Montesinos, 1996; Miller et al., 2001;
Schaad et al., 2001; Singh et al., 2000; Timmusk et al., 1999;
Valdenegro et al., 2001; Wilkinson et al., 1989; Zehnder et
al., 2001). Orchid-associated bacteria have been shown to
follow a seasonal pattern of abundance and differed between
orchid genera (Wilkinson et al., 1994). The objective of this
study was to isolate and identify pathogenic and nonpathogenic bacteria associated with necrotic foliar spots in
Revista Mexicana de FITOPATOLOGIA/
339
cultured plants of L. aromatica collected in three different
environments.
MATERIALS AND METHODS
Collection of leaf samples. Leaf samples of Lycaste
aromatica were collected from August to October 2002 from
three plants from Comitan, Chiapas; four from Orizaba,
Veracruz; and three from Cuetzalan, Puebla, Mexico, in their
native localities. One or two days later, plants were placed
in the greenhouse.
Isolation and characterization of bacteria. With a sterile
dissecting needle, small tissue portions (1-3 mm) that included
live and necrotic tissue were slightly macerated in a sterile
Petri dish. These preparations were then spread on the surface
of King’s B medium (Schaad et al., 2001), in which the
protease peptone was substituted by polypeptone. Three tissue
samples per plant were left in the different Petri plates and
incubated at 25-33°C. All tissue samples were taken from
necrotic spots with yellow halo. From these cultures different
colonies were isolated and pure cultures were obtained. The
isolates were labeled according to the origin. In order to
identify the isolates to the genus level, the methodology
described by Schaad et al. (2001) was used following the
basic tests: Gram strain, production of fluorescent pigments
in King’s B medium, aerobic and anaerobic growth in Hugh
and Leifson’s medium, oxidase and catalase activity, potato
rot, hypersensitivity reaction in tobacco (Nicotiana tabacum
L.) leaves, production of indol, bacterial morphology, and
presence and distribution of flagella. The pathogenicity tests
were performed on plants grown in a greenhouse. A small
injury was produced on the leaf surface with a sterile needle,
and a small piece of cotton fiber impregnated with an aqueous
bacterial suspension (108 cfu/ml) which was placed on the
injury for one day. One bacterial isolate per leaf was tested.
Due to the few plants available, leaves were dissected from
their plants five minutes after they were inoculated, and placed
in a glass with sterile water during fifteen days. With this
information, taxonomic groups were differentiated, and when
necessary, specific tests were added for each group of bacteria,
such as: levan production, arginine dihydrolase, endospores
formation, colony color and growth on yeast extract-dextroseCaCO3 medium (YDC), utilization of carbon sources, and
growth on potato infusion medium (PI) under anaerobic
environment. The anaerobic condition was obtained in a
Gaspak 150 jar with an alcohol lamp inside. The results from
this method were compared with the obtained from bacteria
cultivated into a nalgene® vacuum desiccator which was
tightly sealed. The air was exhausted from the desiccators
with a vacuum pump (Gast, model 522) and after the 700 ml
of mercury vacuum condition was stabilized; the desiccator’s
valve was tightly sealed. The desiccators containing the
bacterial cultures were kept in the incubator at 26-28°C. Both
methods included a culture of an isolate of Xanthomonas as
control.
Fig. 1. Orquid (Lycaste aromatica) leaf depicting typical
necrotic areas caused by bacteria.
RESULTS AND DISCUSSION
Characterization of bacteria. A total of 42 isolates were
obtained from leaves of 10 plants of L. aromatica, 27 from
plants from Cordoba, 6 from Comitan, and 9 from Cuetzalan.
Figure 1 shows leaf necrotic areas from which bacteria were
isolated. Based on the characterization, isolates were
classified into six groups (Table 1). Isolates from the same
location and with the same results on the biochemical tests
were considered individually, because they were obtained
from different plant or leaf; it does not necessary mean that
they are genetically different. Thirty eight percent of the
isolates belonged to the genus Bacillus, 35.8% to the
Enterobacteriaceae, 7% to Pseudomonas, 4.8% to
Xanthomonas, 2.3% to Clostridium, and 12% to bacteria with
coccus morphology. The 16 isolates of the genus Bacillus
(Table 1) were identified as B. circulans Jordan, B. anthrasis
Cohn, B. cereus Frankland and Frankland, B. licheniformis
(Weigmann) Chester, B. coagulans Hammer, B. alvei Cheshire
and Cheyne, B. laterosporus Laubach or B. macerans
Schardinger (Schaad et al., 2001). The 15 isolates of
Enterobacteriace did not show pectinolytic activity and with
the exception of isolate P37, all had oxidase activity. Thirteen
of these isolates probably belong to the Amylovora group
(Burrill) Winslow, Broadhurst, Buchanan, Krumwiede,
Rogers and Smith group, and 2 isolates to Pantoea, because
they had yellow colonies on YDC medium. The Pseudomonas
isolates were aerobic and non pathogenic; therefore, the
belong to the saprophytic Pseudomonas group. On the basis
of tests such as of levan production, oxidase activity,
pectinolytic capacity, production of argenine dihydrolase,
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/ Volumen 22, Número 3, 2004
Table 1. Characterization of bacterial isolates obtained from Lycaste aromatica leaves after morphological, physical, and
biochemical tests.
Isolates
V7
V10
V2
V4
V16
V18
V26
V28
P30
P34
P37
C39
C40
C41
C42
V22
C23
C26
V12
V14
V3
V5
V6
V8
V9
V14
V19
V20
V25
V27
V29
P31
P32
P35
P38
V11
V1
V15
V17
V21
P33
P36
Gram O/F
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+/+/+/+/+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
-/+
+/+
+/+
+/+
+/+
+/+
+/+
Fluor
Morphology
+
+
+
-
bacilliform
bacilliform
bacilliform
bacilliform
bacilliform
bacilliform
bacilliform
bacilliform
bacilliform
bacilliform
bacilliform
bacilliform
bacilliform
bacilliform
bacilliform
bacilliform
bacilliform
bacilliform
bacilliform
bacilliform
bacilliform
bacilliform
bacilliform
bacilliform
bacilliform
bacilliform
bacilliform
bacilliform
bacilliform
bacilliform
bacilliform
bacilliform
bacilliform
bacilliform
bacilliform
bacilliform
coccus
coccus
coccus
coccus
coccus
coccus
Flagella
Peritri
Peritri
Peritri
Peritri
Peritri
Peritri
Peritri
Peritri
Peritri
Peritri
Peritri
Peritri
Peritri
Peritri
Peritri
1-4 polar
1-4 polar
1-4 polar
1 polar
1 polar
Test
L O P A T Indol YYDC MYDC Ana End
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
- +
- +
- +
-
-
-
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Groups
Enterobacteriaceae
Enterobacteriaceae
Enterobacteriaceae
Enterobacteriaceae
Enterobacteriaceae
Enterobacteriaceae
Enterobacteriaceae
Enterobacteriaceae
Enterobacteriaceae
Enterobacteriaceae
Enterobacteriaceae
Enterobacteriaceae
Enterobacteriaceae
Enterobacteriaceae
Enterobacteriaceae
P. fluorescens
P. fluorescens
P. fluorescens
Xanthomonas
Xanthomonas
Bacillus
Bacillus
Bacillus
Bacillus
Bacillus
Bacillus
Bacillus
Bacillus
Bacillus
Bacillus
Bacillus
Bacillus
Bacillus
Bacillus
Bacillus
Clostridium
coccus
coccus
coccus
coccus
coccus
coccus
O/F = Oxidative/fermentative; Fluor = Fluorescent; L = Levan production; O = Oxidase activity; P = Pectinolytic; T =
Hypersensitivity in tobacco (Nicotiana tabacum); I = Indol production; YYDC = Yellow colonies on YDC medium; MYDC =
Mucoid colonies on YDC medium; Ana = Growth in anaerobic environment; End = Endospores; A = Use of argentine.
hypersensitivity reaction on tobacco (Nicotiana tabacum L.)
and the utilization of carbon sources, the three isolates of
Pseudomonas are saprophytes of P. fluorescens Migula
(Schaad et al., 2001). The isolates that belong to the genus
Xanthomonas, showed yellow mucoid colonies on YDC
medium at 30°C. In the case of the isolate characterized as
Clostridium, it grew in an anaerobic condition, and did not
show pectinolytic activity. In this test, the isolate of
Xanthomonas used as control did not grow under these
conditions.
Distribution of the isolates by genus. Isolates varied
depending on the location from where leaves of L. aromatica
were obtained (Fig. 2). The isolates of the genus Bacillus
and the Enterobacteriaceae group were predominant. It is
noticeable the diversity of the bacterial populations from
Cordoba. This could be explained by the environmental
342
/ Volumen 22, Número 3, 2004
as saprophytes or as specific associated of L. aromatica.
Bacteria associated specifically with plants, and that are
pathogenic or beneficial, generally present bacilliform
morphology, whereas bacteria with coccid cellular
morphology are primarily associated with animals and soil
(Lim, 1998; Schaad et al., 2001), so, they can be considered
as casual. The three isolates which correspond to
Xanthomonas and Clostridium were also considered as casual,
due to its low frequency of occurrence and because they did
not originate any pathogenic or hypersensitive response on
leaves of L. aromatica or tobacco. More over, although the
genus Clostridium includes saprophytic species, these require
anoxic or microoxygenic conditions in order to degrade plant
tissue (Schaad et al., 2001). None of the isolates resulted
positive for total indol production, which indicates that they
do not produce auxins which may influence plant growth,
such as indol acetic acid and indolbutyric acid (Barazani and
Friedman, 1999). None of the isolates produced a
hypersensitivity response in tobacco or L. aromatica, which
is the first step in the systemic resistance induction. Bacillus,
Enterobacteriaceae, and Pseudomonas bacteria can promote
plant growth through other mechanisms. There is no
information that would indicate that the genera Xanthomonas
and Clostridium would promote plant growth. The genus
Bacillus includes species that produce antibiotics that have
an effect against fungi, bacteria, and algae. Silo-Suh et al.
(1998) demonstrated that B. cereus produce the antibiotic
zwittermycin A, which has an effect against Oomycetes and
related pathogens, against algae, and bacteria. Hyronimus et
al. (1998) demonstrated that B. coagulans produce the
bacteriocin coaguline. This may be beneficial to the plant as
is the case of Agrobacterium vitis (Ophel and Kerr) Young,
Kuykendall, Martínez-Romero, Kerr and Sawada, whose
development and pathogenic action is interfered through
bacteriocin produced by a nonpathogenic Agrobacterium sp.
(Herlache and Triplett, 2002). Bacillus sp. also produce toxic
compounds which have effects on insects such as Dipterae
(Nicolas, 1992), Coleopterae (Augustin et al., 1998), and
Lepidopterae (Chilcutt and Tabashnik, 1997). Bacillus also
affects fungi, as demonstrated by Fucikovsky et al. (1989) in
which, different species of Bacillus lysed the mycelium of
Curvularia lunata (Wakk.) Boedijn and killed other fungi.
In the case of Enterobacteriaceae, these produce antagonistic
compounds against other microorganisms. Reeves et al.
(1993), reported that E. carotovora subsp. carotovora Jones
produce an extracellular enzyme which acts against E.
chrysanthemi Burkholder, McFadden, and Dimock,
Klebsiella oxytoca (Flügge) Lautrop, Aeromonas hydrophila
(Chester) Stainer, P. aeruginosa (Schroeter) Migula, and X.
campestris (Pammel) Dowson. Fernandes and Marcelo
(2002) reported that strains of the genus Erwinia limited the
development of P. syringae pv. savastanoi (Smith) Young,
Kuykendall, Martínez-Romero, Kerr and Sawada. Other
mechanism by which Erwinia competes with other
microorganisms is through the production of siderophores
(Montesinos, 1996). Different species of Pseudomonas
produce antibiotics such as bacteriocin (Padilla et al., 2002);
the fluorescent Pseudomonas produce siderophores, which
interfere mainly in fungal development (Kurek and JaroszukScisel, 2003) and they can utilize the siderophores produced
by other microorganisms (Loper and Henkels, 1999). In the
case of P. fluorescens, this bacterium was found to be
associated with mycorrhizal fungi of the genus Glomus
(Marschner and Croweley, 1996), and it was also found to be
associated with roots and seeds of orchids as a growth
promoter (Wilkinson et al., 1989).
CONCLUSIONS
None of the bacterial isolates found in L. aromatica leaves
were pathogenic. No plant growth promoting activity based
on production of auxins from tryptophan was detected in these
isolates. No indications of sistemyc induced resistance were
found. Microorganisms of the genus Bacillus,
Enterobacteriaceae, and P. fluorescens, express efficient
mechanisms to interfere the development of other
microorganisms, which may explain their prevalence on the
leaves of L. aromatica.
Acknowledgements. To CONACyT (Mexico) for the
scholarship 135877 for Rodrigo Gabino-Gutiérrez.
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