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© 2005 Plant Management Network.
Accepted for publication 17 May 2005. Published 19 July 2005.


Differentiation of Isolates of Glomerella cingulata and Colletotrichum spp. Associated with Glomerella Leaf Spot and Bitter Rot of Apples Using Growth Rate, Response to Temperature, and Benomyl Sensitivity


Eugenia González and Turner B. Sutton, Department of Plant Pathology, North Carolina State University, Raleigh 27695


Corresponding author: Turner B. Sutton. turner_sutton@ncsu.edu


González, E., and Sutton, T. B. 2005. Differentiation of isolates of Glomerella cingulata and Colletotrichum spp. associated with Glomerella leaf spot and bitter rot of apples using growth rate, response to temperature, and benomyl sensitivity. Online. Plant Health Progress doi:10.1094/PHP-2005-0719-01-RS.


Abstract

Cultural characteristics were investigated as a way to distinguish isolates of Glomerella cingulata and Colletotrichum spp. associated with Glomerella leaf spot and bitter rot of apples from those that cause only bitter rot. The growth rate, response to temperature, and benomyl sensitivity of 27 isolates of Glomerella cingulata, 12 isolates of Colletotrichum gloeosporioides, and 7 isolates of C. acutatum, collected from apple orchards located in the U.S. and Brazil and previously characterized based on morphology, vegetative compatibility, and mitochondrial DNA (mtDNA) haplotypes, were determined. These isolates represent the genetic and molecular diversity within isolates of C. gloeosporioides, C. acutatum, and G. cingulata from apples found in a previous study. Slower growth, lower optimum growth temperature, and less sensitivity to benomyl distinguished isolates of C. acutatum from isolates of G. cingulata and C. gloeosporioides. However, growth rate and benomyl sensitivity were not useful for distinguishing between G. cingulata and C. gloeosporioides or differentiating isolates of G. cingulata that cause leaf spot and bitter rot from those that only cause bitter rot.


Introduction

Fig 1. Bitter rot disease of apples (Colletotrichum gloeosporioides, C. acutatum, and Glomerella cingulata).

 

Bitter rot is a common fruit disease of apples in practically all countries where they are commercially grown. In moist and temperate growing regions it is considered one of the most important diseases and it can cause crop losses as great as 50% (16). Three taxa, Colletotrichum gloeosporioides (Penz.) Penz. & Sacc., C. acutatum J. H. Simmonds, and Glomerella cingulata (Stonem.) Spauld. & Schrenk, are associated with the bitter rot disease of apples (16) (Fig. 1). In 1988, Leite et al. (13) described a new apple leaf spot disease on the cultivars Gala and Golden Delicious in Paraná State in Brazil and associated it with homothallic isolates of C. gloeosporioides, which are designated G. cingulata (14). These isolates produce fertile perithecia and are often referred to as perithecial isolates (15). This was the first report of Colletotrichum spp. causing a leaf spot on apples. The disease, named Glomerella leaf spot (GLS), is characterized by small irregular necrotic spots on the leaves (Fig. 2). It has increased in severity and has become a great concern to Brazilian apple growers, because one of the most widely grown cultivars, Gala, is highly susceptible to the disease. Under favorable conditions GLS can result in 75% defoliation (Fig. 3) by harvest, weakening apple trees and reducing yield (4,13,17). Additionally, GLS has been observed on other commercially important cultivars grown in Brazil, such as Granny Smith and Pink Lady (Dr. Rosa Maria Sanhueza, personal communication, 2002). GLS was first reported in the U.S. in 1998 as a severe leaf spot on cv. Gala apples in two orchards in eastern Tennessee (9) and has subsequently been reported from Georgia and North Carolina. Although GLS was first reported in the U.S. in 1998, experiments conducted in Georgia in the late 1960s (19) with an isolate of G. cingulata suggest that strains of the fungus capable of causing leaf spot were present much earlier.


     
 

Fig 2. Glomerella leaf spot (Glomerella cingulata) on apples, characterized by irregular leaf spot.

 

Fig 3. Defoliation in apple trees caused by GLS. Photo courtesy of Dr. Rosa Maria Sanhueza.

 

Although both GLS and bitter rot are associated with the same fungus, differences in morphology, cultural characteristics, and pathogenicity between isolates of the pathogen obtained from fruit or leaves have been observed (13,19). These differences coupled with the considerable genetic and molecular variability within isolates of C. gloeosporioides and G. cingulata obtained from fruit with bitter rot symptoms (3,15) and within populations of C. gloeosporioides and G. cingulata from other crops (5,6,7,11,20,21) suggest that the diversity of these pathogens on apples is high. A recent study that involved morphological, genetic, and molecular characterization of isolates of G. cingulata and C. acutatum, and C. gloeosporioides obtained from apples collected in different locations in the U.S. and Brazil, showed a high diversity within each species (8). Twelve morphological types, 16 VCGs, 12 mtDNA haplotypes, and 7 phylogenetic groups, summarized in Table 1, were found among the isolates. Additionally, foliar pathogenicity tests showed that only isolates of G. cingulata with specific VCGs and haplotypes were pathogenic to leaves (Table 1).


Table 1. Characterization and pathogenicity of isolates of G. cingulata, C. gloeosporioides, and C. acutatum according to morphological, genetic, and molecular groups found among isolates collected in apple orchards located in the U.S. and Brazilx.

Species Sequence
analysis
mtDNA
haplo-
type
VCG Morpho-
logical
type
Leaf
patho-
genicity
Location
G.
cingulata
Group 1 G1 1 SP1 + U.S.
1 SP1 + U.S.
2 SP1,SP2 - U.S.
3 SP1 - U.S.
G1.1 1 SP1 + U.S.
1 SP1 - U.S.
2 SP2 - U.S.
G2 2 SP2 - U.S.
G2.1 2 SP2 - U.S.
Group 2 G3 4 SP1 + Brazil
5 SP1 + Brazil
G4 5 SP1 + Brazil
Group 3 A3 6 CP - U.S.
A3.1 6 SP3 - U.S.
C. gloeo-
sporioides
Group 4 B2 7 SS2 - U.S.
12 SS5 - U.S.
Group 5 B2 n/a     - U.S.
B3 8 SS3 - U.S.
9 SS1 - U.S.
Group 6 B2 11 SS3 - U.S.
B3 11 SS3 - U.S.
Group 7 B2 10 SS1 - U.S.
C.
acutatum
n/a C1 15 SSC - U.S.
n/a 16 SSNC n/a U.S.
n/a 13 SSNC(O) n/a Brazil
D1 14 SSNC(O) - Brazil

 x Sequence analysis (groups based on Maximum Likelihood and Maximum Parsimony phylogenetic trees based on the sequence of a 200 bp intron of the GDPH nuclear gene), mtDNA haplotypes (mtDNA RFLPs of genomic DNA digested with MspI), VCG (vegetative compatibility groups), morphological types (based on colony color, conidial shape, the ability to produce perithecia in culture, and distribution of acervuli and perithecia in culture), and pathogenicity (determined by inoculating trees of cv. Gala grown under greenhouse conditions) were described in a previous study (8). ‘+’ indicates pathogenic; ‘-’ indicates not pathogenic. n/a = data not available.


Cultural characteristics have also been used to distinguish among isolates of C. gloeosporioides, G. cingulata, and C. acutatum. Isolates of G. cingulata and C. gloeosporioides are characterized by a faster growth rate (10,12,14,18) and a greater sensitivity to benomyl (1,2,12,18). Additionally, isolates of C. acutatum have shown lower optimum growth temperatures than isolates of C. gloeosporioides (1). Genetic and molecular tools can be time-consuming and expensive, therefore, the use of cultural characteristics, may provide a quicker and less expensive way to differentiate isolates of G. cingulata associated with GLS.

The objective of this study was to use growth rate, response to temperature, and benomyl sensitivity to determine if there are differences among mtDNA haplotypes of C. gloeosporioides, C. acutatum, and G. cingulata and, if there are differences, determine if they can be used to distinguished isolates of G. cingulata associated with GLS from those that cause bitter rot only.


Cultural Characterization of Isolates

Twenty-seven isolates of G. cingulata (14 from fruit and 13 from leaves), 12 fruit isolates of C. gloeosporioides, and 7 isolates of C. acutatum (four from fruit and three from leaves) were selected from the collection of monosporic isolates, previously characterized based on morphology, vegetative compatibility, and mtDNA RFLP haplotypes (8) (Table 2) to determine growth rate, optimum growth temperature, and sensitivity of the isolates to benomyl. The isolates represented mtDNA haplotypes A3, B2, B3, C1, D1, G1, G2, G3, and G4. Leaf spot isolates of G. cingulata are found within haplotypes G1, G3, and G4. The growth rate (in mm/day) of isolates of G. cingulata, C. gloeosporioides, and C. acutatum was determined by measuring the colony diameter of isolates every 24 h for 6 days at 25ºC with constant light. Petri dishes containing approximately 15 ml of PDA were inoculated with 5-mm-diameter plugs of each isolate, obtained from the margins of 4-day old cultures. Three Petri dishes were used for each isolate and the experiment was repeated once. Sensitivity to benomyl was determined by amending Petri dishes containing 15 ml of PDA with each of the following concentrations: 0, 0.01, 0.1, 1 and 10 µg/ml. Three Petri dishes were used per isolate and two runs of the experiment were conducted.


Table 2. Isolates of G. cingulata, C. gloeosporioides, and C. acutatum examined for growth rate, response to temperature and sensitivity to benomylx.

Species Isolate
desig-
nation
Geo-
graph-
ical
origin
y
Source mtDNA
haplo-
type
VCG Morph-
ological
type
Host
tissue
cv.z
G.
cingulata
GA(L) 13 GA Leaf Gala G1 1 SP1
GA 16 GA Fruit Gala G1 1 SP1
CROTTS(L) 27 NC Leaf Gala G1 1 SP1
TN 7 TN Leaf Gala G1 1 SP1
LD 11 NC Fruit GS G1 2 SP1
LD 16 NC Fruit GS G1 2 SP1
LD 25 NC Fruit GS G1 2 SP1
LD 20 NC Fruit GS G1 2 SP1
OH 3 OH Fruit MD G1 3 SP1
CROTTS 1 NC Fruit Gala G2 2 SP1
CROTTS 2 NC Fruit Gala G2 2 SP1
CROTTS 3 NC Fruit Gala G2 2 SP1
CROTTS 5 NC Fruit Gala G2 2 SP1
BR 2 Brazil Leaf Gala G3 5 SP1
BR 3 Brazil Leaf Gala G3 4 SP1
BR 8 Brazil Leaf Gala G3 4 SP1
BR 10 Brazil Leaf Gala G3 4 SP1
BR 13 Brazil Leaf Gala G3 5 SP1
BR 17 Brazil Leaf Gala G3 5 SP1
BR 19 Brazil Leaf Gala G3 5 SP1
BR 9 Brazil Leaf Gala G4 5 SP1
BR 21 Brazil Leaf Gala G4 5 SP1
RD 1 NC Fruit Delicious A3 6 CP
RD 2 NC Fruit Delicious A3 6 CP
LD 2 NC Fruit GS A3 6 CP
LD 12 NC Fruit GS A3 6 CP
TN 8 TN Leaf Gala A3 6 CP
C. gloeo-
sporioides
AL 5 AL Fruit GD B2 7 SS2
AL 6 AL Fruit GD B2 7 SS2
AL 8 AL Fruit GD B2 7 SS2
AL 9 AL Fruit GD B2 7 SS2
AL 1 AL Fruit GD B3 8 SS3
AL 2 AL Fruit GD B3 8 SS3
AL 4 AL Fruit GD B3 8 SS3
AL 10 AL Fruit GD B3 8 SS3
LD Cg 1 NC Fruit GS B3 9 SS1
LD Cg 8 NC Fruit GS B3 9 SS1
LD Cg 11 NC Fruit GS B3 9 SS1
LD Cg 13 NC Fruit GS B3 9 SS1
C.
acutatum
LD Ca 5 NC Fruit GS C1 n/a SSC
LD Ca 10 NC Fruit GS C1 n/a SSC
LD Ca(b) 4 NC Fruit GS C1 n/a SSNC
LD Ca(b) 6 NC Fruit GS C1 n/a SSNC
BR Ca 4 Brazil Leaf Gala D1 13 SSNC(O)
BR Ca 3 Brazil Leaf Gala D1 n/a SSNC(O)
BR Ca 6 Brazil Leaf Gala D1 13 SSNC(O)

 x mtDNA haplotypes, vegetative compatibility groups (VCG), and morphological types were described in a previous study (8).

 y Abbreviations: GA, NC, TN, OH, and AL = Georgia, North Carolina, Tennessee, Ohio, and Alabama, respectively.

 z GS = Granny Smith, MD = Molly’s Delicious, GD = Golden Delicious.


To determine optimum growth temperature, 5-mm-diameter plugs from the margins of 4-day-old cultures of the isolates of G. cingulata, C. gloeosporioides, and C. acutatum were placed in the center of Petri dishes containing 15 ml of PDA medium and incubated in the dark at 14, 18, 24, 26, and 30°C. Five-mm-diameter plugs of each isolate were placed on three different PDA dishes, and colony diameter was measured after 2, 4, and 6 days. The experiment was repeated once.


Data Analyses

The optimum temperature for the growth of each species was estimated by fitting a quadratic equation to the growth of each species at all temperatures tested [growth = b0 + b1(temp) + b2(temp2)] and solving the following equation: maximum growth = (-b1/2b2), where b1 and b2 are the coefficients for the linear and quadratic terms. The reduction in growth of isolates of G. cingulata, C. gloeosporioides, and C. acutatum at less then optimum temperatures was determined by calculating the percentage reduction in colony diameter compared to the colony diameter at the temperature where maximum growth occurred.

Mean growth within haplotypes and species in each experiment was compared with an analysis of variance using SAS (Windows version, release 6.12; SAS Institute, Inc., Cary, NC). Means were separated by the Waller-Duncan k-ratio t test. The EC50 (effective concentration of benomyl to reduce growth by 50%) of each isolate to benomyl was calculated based on the mean of the colony diameter of isolates within each haplotype and species 6 days after incubation using the Proc Probit log10 program in SAS.


Effect of Temperature and Benomyl on Isolate Growth

Overall, the isolates of G. cingulata and C. gloeosporioides grew faster and were more sensitive to benomyl than isolates of C. acutatum (Table 3). There were no significant differences in growth rate and benomyl sensitivity between isolates of G. cingulata and C. gloeosporioides. The rate of growth between isolates of C. acutatum collected from the U.S. (C1) and Brazil (D1), was significantly different, and isolates of C. acutatum from the U.S. grew slower than all of the haplotypes tested.


Table 3. Growth rate and benomyl sensitivity of isolates of G. cingulata, C. gloeosporioides, and C. acutatum

Species No. of
isolates
mtDNA
haplo-
type
v
Growth (mm/day)wy EC50xy
Haplo-
type
mean
Species
mean
Haplo-
type
mean
Species
mean
G. cingulata 5 A3 13.0 a   0.19 c     
G. cingulata 7 G3 12.1 c   0.12 dc  
G. cingulata 4 G2   12.4 ba   0.10 dc  
G. cingulata 2 G4   12.3 bc   - z  
G. cingulata 9 G1 12.2 c 12.4 a 0.15 dc 0.14 b
C. gloeo-
sporioides
4 B2   12.9 ba   0.16 dc  
C. gloeo-
sporioides
8 B3 11.4 d 11.9 a 0.07 d 0.10 b
C. acutatum 4 C1  9.2 e   0.37 b  
C. acutatum 3 D1  8.5 f   8.9 b 0.66 a 0.47 a

 v mtDNA haplotypes described in a previous study (8).

 w Growth represents the colony diameter (mm/day) of the isolates at 26°C over 6 days.

 x EC50s were calculated using Proc Probit log 10 in SAS, based on the colony diameter of the isolates at 0, 0.01, 0.1, 1, and 10 m g/ml of benomyl after 6 days of incubation at 26°C.

 y Means followed by the same letter are not significantly different at P = 0.05 according to the Waller-Duncan k-ratio t test.

 z Haplotype was not examined.


C. acutatum had higher EC50s to benomyl than isolates of G. cingulata and C. gloeosporioides. Isolates of C. acutatum from Brazil were the least sensitive with an EC50 of 0.66 mg of benomyl per ml (Table 3).

The predicted optimum temperatures for the growth of isolates of G. cingulata, C. gloeosporioides, and C. acutatum were 24.0, 24.2, and 22.0°C, respectively; 14°C was the least favorable temperature for all species and growth at this temperature was reduced by 68 to 72% compared to the optimum (Fig. 4). The greatest differences in growth among species occurred at 30°C (Fig 5). At 30°C, isolates of C. acutatum grew significantly slower than isolates of G. cingulata and C. gloeosporioides. At this same temperature, differences within isolates of the same species were also observed. Within C. gloeosporioides B3 isolates grew slower than B2 isolates. Isolates of G. cingulata with haplotypes G1 and G2 grew slower than the ones with haplotypes G3, G4, and A3.


   
 

Fig 4. Effect of temperature on the growth of isolates of G. cingulata (A), C. gloeosporioides (B), and C. acutatum (C). Maximum growth = (-b1/2b2), where b1 and b2 are the coefficients for the linear and quadratic terms, respectively, in the quadratic equation [growth = b0 + b1(temp) + b2(temp2)]. Maximum growth reduction was calculated at 14°C for the three species.

 

 

Fig 5. Growth of isolates of G. cingulata, C. gloeosporioides, and C. acutatum at five different temperatures (14, 18, 22, 26, and 30°C). Growth represents the average over 6 days of the mean colony diameter (mm/day) of the isolates measured every 2 over 6 days. Isolates tested represent 9 haplotypes found in the mtDNA RFLP analysis and each haplotype is represented by isolates from different locations and apple orchards, if applicable (Table 2).

 

Conclusion

In previous studies, isolates of G. cingulata and C. gloeosporioides were distinguished by their faster growth rate (10,12,14,18) and greater sensitivity to benomyl (1,2,12,18) from isolates of C. acutatum obtained from apples, citrus, strawberry, peach, and other hosts. Additionally, Adaskaveg and Hartin (1) observed a lower optimum growth temperature for isolates of C. acutatum obtained from strawberry, almond, and peach compared to the optimum growth temperature for isolates of C. gloeosporioides obtained from citrus and papaya. However, Gunnell and Gubler (10) stated that isolates of G. cingulata, C. gloeosporioides, and C. acutatum from strawberry had the same optimum growth temperature. In this study, slower growth, less sensitivity to benomyl and a lower optimum growth temperature differentiated isolates of C. acutatum from isolates of G. cingulata and C. gloeosporioides. Growth rate and benomyl sensitivity were not useful for distinguishing between isolates of G. cingulata and C. gloeosporioides or within mtDNA haplotypes, vegetative compatibility groups (VCGs), or morphological groups of each species. Differences among the growth of haplotypes of the same species were observed only at 30°C; however, only isolates of G. cingulata that cause bitter rot and GLS within haplotype G1 were distinguished from isolates of G. cingulata that cause bitter rot only. These results suggest that although cultural characteristics are helpful to differentiate Colletotrichum species from apple, they are not useful for separating isolates below the species level.


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