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Growth and aggressiveness factors affecting Monilinia spp. survival peaches
International Journal of Food Microbiology 224 (2016) 22–27
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Growth and aggressiveness factors affecting Monilinia spp. survival peaches M. Villarino, P. Melgarejo, A. De Cal ⁎ Department of Plant Protection, INIA, Carretera de La Coruña km 7, 28040, Madrid, Spain
a r t i c l e
i n f o
Available online 15 February 2016 Keywords: M. laxa M. fructigena Occurrence of species Lesion lengthstroma Sclerotia
a b s t r a c t Brown rot of stone fruit is caused by three species of Monilinia, Monilinia laxa, M. fructigena, and M. fructicola. Eleven components of 20 different isolates of each of the three Monilinia species were analysed to determine distinct aggressiveness and growth characteristics among the three fungi. M. fructicola showed the greatest lesion diameter, and the lowest incubation and latency period on fruit postharvest, however isolates of M. fructigena exhibited less aggressiveness components. Five growth characteristics of M. fructicola could be used to distinguish M. fructicola from the other two species. The dendrogram generated from only the presence of sclerotia and lesion length on infected fruit separated the 60 isolates into two clusters (r = 0.93). One cluster was composed of the M. laxa and M. fructigena isolates and the other cluster comprised the M. fructicola isolates. However, the dendrogram generated based on the presence of stromata and sclerotia in the same colony of the three species when they were grown on potato dextrose agar, and the lesion diameter on fruit infected with each species separated the 60 isolates into three clusters (r = 0.81). Each cluster comprised the isolates of each of three Monilinia spp. We discussed the effect of M. fructicola growth and aggressiveness differences on the displacement of M. laxa and M. fructigena by M. fructicola recorded in Spanish peach orchards and their effect on brown rot at postharvest. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Brown rot caused by Monilinia laxa (Aderh et Rulh) Honey, M. fructicola (Wint.) Honey, and M. fructigena Honey in Whetzel (Byrde and Willetts, 1977), is an economically important disease of commercial stone fruit production and storage. These three brown rot fungi can infect both the fruit, causing substantial preharvest and postharvest losses, and the blossoms and twigs of stone fruit trees, causing blossom and twig blight (Byrde and Willetts, 1977). Postharvest losses are typically more severe than preharvest losses, and routinely occur during storage and transport, in some cases even affecting fruit at the processing stage (Hong et al., 1997). Until 2006, peach brown rot in Spain was caused either by M. laxa or M. fructigena only (De Cal and Melgarejo, 1999; Gell et al., 2009), with M. laxa the most prevalent (85–90%) (Larena et al., 2005). In 2006, M. fructicola was detected in peach orchards in the Ebro Valley, Lleida, Spain (De Cal et al., 2009). Since 2010, M. fructicola has displaced M. fructigena and co-existed with M. laxa in the similar relative frequency as M. laxa in three Ebro Valley peach orchards (Villarino et al., 2013). Six years after its first detection, the relative frequency of M. fructicola ⁎ Corresponding author at: Plant Protection Department, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Ctra. de la Coruña km 7, 28040, Madrid, Spain. E-mail address: [email protected] (A. De Cal).
http://dx.doi.org/10.1016/j.ijfoodmicro.2016.02.011 0168-1605/© 2016 Elsevier B.V. All rights reserved.
(absolute frequency normalized) increased on stored fruit, latent infections, blighted shoots, pruned branches, and mummified fruit on the trees (Villarino et al., 2013). However, the relative frequency of M. laxa in commercial orchards decreased progressively, especially on harvested fruit (Villarino et al., 2013). Co-existence between M. fructicola and M. laxa has been previously reported in Michigan and California (USA), where the two pathogens have somewhat different niches at early or late-season (Boehm et al., 2001), although M. fructicola is the most prevalent brown rot species in these states of USA. M. fructicola can be distinguished from M. laxa by growth characteristics such as colony shape, colour of its conidia, absence of hyphal anastomoses between germinating conidia, germ tube extension of the conidia before germ tube branching, and also by VCG (De Cal and Melgarejo, 1999; Mordue, 1979a, 1979b; Penrose et al., 1976). M. fructicola produces conidia and spermatia more abundantly than M. fructigena, and its conidia size and hyphal diameter are smaller than those of M. fructigena (Mordue, 1979a, 1979c). M. fructigena can be distinguished from M. laxa by its colony margins, the different conidial colour, and the length of conidial germ tubes (De Cal and Melgarejo, 1999; Mordue, 1979b, 1979c). Microconidia can be found on decaying fruit infected with M. fructicola and in cultures of M. laxa and M. fructicola which are older than 30 days (Ogawa et al., 1975). Irregular stromatal crusts and discoid sclerotia may develop on the agar surface or within the medium as colonies age. Of the three fungi, the apothecia of M. fructicola can be produced from pseudosclerotial mummified fruit
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under field (Biggs and Northover, 1985; Byrde and Willetts, 1977; Holtz et al., 1998) or laboratory conditions (Baxter et al., 1974; De Cal et al., 2014; Holtz et al., 1998; Willetts and Harada, 1984). In contrast, the apothecia of M. fructigena and M. laxa are not found in the field (Janisiewicz et al., 2013; Villarino et al., 2010) and have not yet been produced under laboratory conditions (De Cal et al., 2014; Willetts and Harada, 1984). Infection efficiency, latent period, spore production rate, infection period, and lesion size were considered as components of the pathogen aggressiveness, although these characteristics could vary between isolates within a species (Pariaud et al., 2009). Infection and sporulation of Monilinia spp. could also be influenced by the saprophytic and pathogenic fitness of each species and climatic conditions. The main objective of this study was to compare the growth and aggressiveness factors of the three Monilinia spp. in culture media and on infected fruit at postharvest, in order to identify any features which could be related to their survival and to the displacement of M. laxa and M. fructigena by M. fructicola in Spanish peach orchards. 2. Materials and methods 2.1. Fungal isolates Sixty isolates (20 each of M. fructicola, M. fructigena, and M. laxa) from different stone fruit from across the world were studied. The host, geographical origin, year, and number of isolates are listed in Table 1. Isolation and culturing of the 60 isolates was done on potato dextrose agar (PDA) (Difco Laboratories) supplemented with 0.5 g streptomycin sulphate/l (Lab. Reig Jofré S.A., Barcelona, Spain). Forty-
Table 1 Geographical origin, host, year, and number of Monilinia isolates. Specie
Geographic origin
M. fructicola
USA Bellegarde, Balandrán, France Albesa, Lleida, Spain Alfarràs, Lleida, Spain Sudanell, Lleida, Spain Lleida, Spain Alfarràs, Lleida, Spain Lleida, Spain Hamilton, New Zealand M. fructigena Australia Japan Portugal A Coruña, Spain Jubia, A Coruña, Spain Jubia, A Coruña, Spain Lleida, Spain Bellegarde, Balandrán, France Lleida, Spain Albesa, Lleida, Spain Alfarràs, Lleida, Spain Sudanell, Lleida, Spain M. laxa South Africa Cehegin, Murcia, Spain Bellegarde, Balandrán, France Albesa, Lleida, Spain Alfarràs, Lleida, Spain Sudanell, Lleida, Spain Serós, Lleida, Spain Riba-roja d'Ebre, Tarragona, Spain Zaidi, Lleida, Spain Zaidi, Lleida, Spain Lleida, Spain Alfarràs, Lleida, Spain Gimenells, Lleida, Spain Sudanell, Lleida, Spain Riba-roja d'Ebre, Tarragona, Spain Albesa, Lleida, Spain
(–) No information.
Host
Isolation Number year of isolates
Plum Peach Peach Peach Nectarine Nectarine Peach Peach – Prunus spp. Apple Quince Plum Apple Plum Nectarine Peach Peach Peach Peach Nectarine Peach Apricot Peach Peach Peach Nectarine Nectarine Peach Peach Nectarine Peach Peach Peach Nectarine Peach Peach
1994 2001 2006 2006 2006 2006 2007 2007 2008 1971 1995 1996 1996 1996 1996 1998 2001 2006 2006 2006 2006 1996 1996 2001 2006 2006 2006 2006 2006 2006 2006 2007 2007 2007 2007 2007 2008
1 1 1 3 2 1 7 3 1 1 1 2 1 1 1 2 6 1 2 1 1 1 1 1 2 3 1 1 1 1 1 1 1 1 2 1 1
23
four isolates were collected from different cultivars of peaches and nectarines in the Ebro Valley, Spain. Sixteen isolates were sent from France, Portugal, New Zealand, Australia, Japan, and South Africa. The isolates were identified as M. fructicola, M. fructigena, or M. laxa, using their growth characteristics (De Cal and Melgarejo, 1999) and a PCR-based assay (Gell et al., 2007). In order to investigate the 60 isolates in the collection, each isolate was stored at − 80 °C in 20% glycerol (long-term storage) and at 4 °C on PDA slants in the dark (short-term storage). The isolates were grown on PDA at 22–25 °C in the dark for mycelium and spore production. 2.2. Aggressiveness components Aggressiveness of each isolate of all Monilinia spp. was tested on four nectarine fruit, cv. `Big Top´ which is an early variety with high productivity (Badenes et al., 1999). The nectarines were sanitized by immersing each fruit in a 1% NaOCl solution for 5 min, and then in 70% ethanol for 1 min. After two rinses in sterile distilled water (SDW), three puncture wounds were made in the skin of each fruit and 25 μl conidial suspension (104 conidia/ml SDW) were put on each wound. The inoculated fruits were then incubated at 22 ± 2 °C for 7 days under fluorescent lighting (100 μE/m2s with a 16-h photoperiod) in humidity chambers that were lined with moist paper (Guijarro et al., 2008). Each fruit was visually examined daily for symptoms of brown rot, and the incidence of brown rot caused by each Monilinia isolate was recorded (Guijarro et al., 2008). The percentage of fruit with brown rot symptoms (% brown rot incidence), the incubation period (the time interval between infection inoculation and the onset of symptom from that infection) and latency period (the time interval between infection inoculation and the onset of sporulation from that infection) (Pariaud et al., 2009) were recorded for each infected fruit. The daily lesion length (diameter in mm/day) was calculated from the individual measurements of the lesion's diameter on fruit that were made on each day of the 7-day incubation using regression analysis. To determine sporulation, the infected area was first excised from the fruit surface, and then suspended in 100 ml ethanol (70%), sonication for 5 min. This volume was filtered through glass wool, centrifuged at 14,040 g for 10 min at 4 °C (Sorvall RC 5C Plus, GMI) and the resultant pellet was transferred to 5 ml SDW. The number of conidia of Monilinia spp. on brown rot lesion was counted (conidia/cm2 of each infected area) using a hemacytometer and a light microscope. The complete experiment was repeated twice. 2.3. Growth components The six growth components were investigated for all 60 isolates. Two components, namely the percentage germination of conidia (%) and the length of the germ tubes (μm), were studied under light microscopy. Four components, namely colony growth rate (mm/day), in vitro sporulation (number of conidia/cm2), and the absence or presence of stromata, and sclerotia, were studied when the isolates were grown on PDA. 2.3.1. Germination components In order to produce conidia for studying the germination component, the isolates were grown on PDA or PDA that was amended with 1% acetone for seven days in the dark at 22 °C, as recommended by Pascual et al. (1990). Those colonies that did not produce any sporulation were maintained at 5–10 °C for 60 days, as recommended by Masri (1967) and Tamm and Flückiger (1993). The different ways of producing the spores could have influenced the results. Conidia were removed from the surface of the colony with a sterile scalpel, suspended in SDW, and then sonicated for one minute. A 15-μl droplet of the conidial suspension (106 conidia/ml) was spread on 30 μl of Czapek Broth (Difco) on sterilized slides, which were incubated for 24 h at 22 °C
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Table 2 Comparison of the brown rot incidence, diameter of lesion, sporulation, and incubation and latency periods of Monilinia fructicola, M. fructigena, and M. laxa growing on nectarine fruit cv. `Big Top´. Species
Brown rot incidence (%)
Diameter of lesion (mm/day)
Sporulation (number of conidia/cm2)
Incubation period (days)
Latency period (days)
M. fructicola M. fructigena M. laxa MSE
88.1 a 72.2 b 82.1 a 399.8
3.6 a 2.2 c 3.0 b 1.9
0.7 × 106 b 1.1 × 106 b 3.3 × 106 a 1.8 × 1012
3.4 b 3.6 ab 3.7 a 0.2
4.0 b 4.4 a 4.5 a 0.2
Data are displayed as the mean of three wounds per fruit. Data were analysed by analysis of variance using the general linear model procedure in Statgraphics XVI Centurion for Windows 7 (StatPoint, Inc. Herndon, VA, USA). When the F test was significant at P = 0.05, the means were compared by the Student–Newman–Keuls multiple range test. Means followed by the same letter in each column are not significant at P = 0.05. MSE — mean squared error.
in the dark. All slides were dried under heat after incubation at the same time. Percentage germination was determined in 50 randomly selected conidia and germ tube length was measured in 25 randomly selected conidia using a light microscope (×100) (Zeiss Axioskop 2; Carl Zeiss, Inc.) with an ocular micrometre. Conidia were considered to have germinated when the germ tube was longer than the length of the conidia. Three replicates (drops) were made for each isolate and each assay was repeated twice. 2.3.2. Growth components on PDA The colony morphology of each isolate was determined on PDA. Plugs (ø 5 mm) of actively growing mycelia were cut from the margins of a 7-day-old colony of each isolate and placed in the centre of a sterile PDA Petri dish (ø 9 cm). The plates were then incubated for 11 days at 22 ± 2 °C in the dark. The absence or presence of stromata and sclerotia in the colony of each isolate was recorded visually on days 5 and 11 of the 11-day incubation. The daily growth rate (mm/day) was calculated from the individual measurements of colony diameter that were made on each day of the 11-day incubation using regression analysis. Sporulation was calculated after by suspending the entire colony of each isolate on PDA in 100 ml ethanol (70%), and sonicating for 5 min. The suspension was then filtered through glass wool and concentrated by centrifuging (Sorvall® RC 5C Plus, GMI) at 14,040 g for 10 min at 4 °C. The resultant pellet was transferred to 5 ml SDW, and the number of conidia of Monilinia spp. was counted using a cell counting chamber and a light microscope. Five replicate plates were used for each isolate and complete each assay was repeated twice. 2.4. Data analysis Data of in vitro and in vivo components of Monilinia were analysed by analysis of variance using the general linear model (GLM) procedure in Statgraphics XVI Centurion for Windows 7 (StatPoint, Inc. Herndon, VA,
USA). For this purpose, the following equation was used: Zijk ¼ μ þ Si þ IðSÞ jðiÞ þ ekðijÞ where μ is a “grand mean”, Z is the variable (in vitro and in vivo components of Monilinia), S (species) is the fixed factor, I (isolate) is nested to species, and e (error) is a random component due to factors that were not included in the model. When the F test was significant at P b 0.05, the means were compared by the Student–Newman–Keuls multiple range test (Snedecor and Cochram, 1980). Mean square error (MSE) and the marginal contribution of each main effect were considered, but only MSE was shown for measuring the contribution of each variable to the model when it is added in the order listed. The data were also analysed using a numerical taxonomy and multivariate analysis system NTSYS-pc version 2.10b (Exeter Software, Setauket, New York, USA) (Rohlf, 1993). Similarity matrices were constructed from the data using simple matching coefficient (SM) (Sokal and Michener, 1958). Dendrograms from the similarity matrices were generated using the unweighted pair-group method with arithmetic average (UPGMA) by the sequential, agglomerative, hierarchical, and nested (SAHN) clustering programme of NTSYS-pc version 2.10b. The goodness of fit of each cluster analysis was calculated by a cophenetic correlation coefficient (Rohlf and Sokal, 1981). When the value of a cophenetic correlation coefficient was ≥ 0.8, this value means that the data within a cluster are most likely to be highly reliable (Rohlf, 1993). The data of the 20 each M. fructicola isolates, M. fructigena, and M. laxa growing on PDA and on nectarine fruit cv. `Big Top´ were also subjected to principal component analysis (PCA) using the GenAlEx 6.2 software programme (Peakall and Smouse, 2006). 3. Results The incidence of brown rot caused by either the M. fructicola or M. laxa isolates was similar (up to 82%) and significantly higher than that of brown rot caused by M. fructigena isolates (72%) (Table 2). The fastest growth rate was in lesions caused by M. fructicola isolates (3.63 mm/day) and these lesions had the shortest incubation and latent periods (3.4 and 4.0 days, respectively) (Table 2). In contrast, sporulation on the infected fruit was greatest on those fruit inoculated with M. laxa (3 × 106 conidia/cm2). The length of the germ tubes was the greatest (51.56 μm) in M. fructicola isolates (Table 3). However, no differences on the germination percentage were observed among isolates. M. fructicola had the fastest colony growth rate (5.93 mm/day), the largest number of produced conidia (104 conidia/cm2), and the most sclerotia on PDA (Table 3). In contrast, M. laxa colonies showed the most stromata of all the colonies studied (Table 3). The results of the principal component analysis (PCA) revealed that the most important factor that distinguished species was the presence of sclerotia and stromata, followed, in declining order, by the lesion length on infected fruit, the germ tube length, the germination percentage, and the latency period (Fig. 1). Components 1, 2, 3, and 4 explained 29.31, 25.84, 17.94, and 15.93% of the variance, respectively.
Table 3 Comparison of the germination percentage, length of the germ tube, colony growth rate, sporulation, presence of sclerotia, and presence of stromata of Monilinia fructicola, M. fructigena, and M. laxa growing on potato dextrose agar (PDA). Species
Germination percentage (%)
Germ tube length (μm)
Colony growth rate (mm/day)
Presence of sclerotia
Presence of stromata
Sporulation (number of conidia/cm2)
M. fructicola M. fructigena M. laxa MSE
43.0 a 53.0 a 40.6 a 311.1
51.6 a 40.4 b 34.7 b 215.2
5.93 a 5.0 b 4.7 b 4.3
0.7 a 0.1 b 0.1 b 0.1
0.6 b 0.3 c 1.0 a 0.1
10,065.5 a 12.9 b 3.5 b 4.0 × 108
Data are displayed as the mean of four fruit with three replications. Data were analysed by analysis of variance using the general linear model procedure in Statgraphics XVI Centurion for Windows 7 (StatPoint, Inc. Herndon, VA, USA). When the F test was significant at P = 0.05, the means were compared by the Student–Newman–Keuls multiple range test. Means followed by the same letter in each column are not significant at P = 0.05. MSE — mean squared error.
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The dendrogram generated from the presence of sclerotia in the colonies of the 20 isolates each of M. fructicola, M. fructigena, and M. laxa isolates when they were grown on PDA and the lesion length on fruit infected with each 20 isolates of each M. fructicola, M. fructigena, and M. laxa separated the 60 isolates into two clusters (r = 0.93) (Fig. 2). The clustering of the isolates in the dendrogram was independent of the year of their isolation and the host. However, the dendrogram generated from the presence of stromata and sclerotia in the colonies of the 20 isolates of each M. fructicola, M. fructigena, and M. laxa when they were grown on PDA and the lesion length on fruit infected with each of the 20 isolates of each of M. fructicola, M. fructigena, and M. laxa separated the 60 isolates into three clusters (r = 0.81). Each cluster comprised the isolates of each of three Monilinia spp. (Fig. 3). Fig. 1. Principal component data analysis of the growth and aggressiveness factors from the 20 isolates each of Monilinia fructicola, M. fructigena, and M. laxa growing on potato dextrose agar (PDA) and on nectarine fruit cv. `Big Top´. PCA revealed the most important factor that distinguished species. Components 1, 2, 3 and 4 accounted for 29.31, 25.84, 17.94, and 15.93% of the variance, respectively.
4. Discussion All Monilinia isolates tested were pathogenic on nectarines at postharvest conditions. We found similar incidence of brown rot on infected
Fig. 2. Dendrogram generated from the presence of sclerotia in colonies of the 20 isolates each of Monilinia fructicola, M. fructigena, and M. laxa when they were grown on potato dextrose agar and the lesion length on fruit infected with each of the 60 isolates. The dendrogram was generated using the unweighted pair-group method with arithmetic average (UPGMA) by the sequential, agglomerative, hierarchical, and nested (SAHN) clustering programme of NTSYS-pc version 2.10b after calculating the simple matching (SM) coefficient. The cophenetic coefficient, r = 0.93.
Fig. 3. Dendrogram generated from the presence of sclerotia and stromata in the colonies of the 20 isolates each of Monilinia fructicola, M. fructigena, and M. laxa when they were grown on potato dextrose agar and lesion length on fruit infected with each of the 60 isolates. The dendrogram was generated using the unweighted pair-group method with arithmetic average (UPGMA) by the sequential, agglomerative, hierarchical, and nested (SAHN) clustering programme of NTSYS-pc version 2.10b after calculating the simple matching (SM) coefficient. The cophenetic coefficient, r = 0.81.
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fruit due to M. fructicola and M. laxa. However, isolates of M. fructicola developed the largest lesions, and the lowest incubation and latency period. Lesions, and the lowest incubation and latency period, have also been related to aggressiveness components (Pariaud et al., 2009). Isolates of M. fructigena resulted in less aggressiveness components. These findings indicate that M. fructigena was less aggressive on nectarine fruit than M. fructicola or M. laxa. Although the three pathogenic Monilinia spp. are polytrophs and can infect a wide range of Rosaceae spp., the pathogenic activity of M. fructigena as a cause of fruit rot is mainly confined to pome fruit (Gril et al., 2008). Of the three brown rot fungi, M. fructicola also exhibited the most differences in growth characteristics on media. Dispersal ability of M. fructicola could be considered better than either M. fructigena or M. laxa because it grows faster (Lichtemberg et al., 2014) and sporulates more abundantly (Byrde and Willetts, 1977). However, Hu et al. (2011) evaluated four Monilinia species for colony and lesion growth rates on PDA and peach fruit, and found that on PDA the fastest colony growth rate was observed for M. fructicola but M. laxa was faster in inoculated fruit. We also found five significantly different growth characteristics of M. fructicola from the other two species. M. fructicola showed the highest values in four of them with potential relationship to aggressiveness, length of germ tube, presence of sclerotia, growth rate, and sporulation. A significant relationship has been observed between growth and sporulation of M. fructicola isolates on PDA and their aggressiveness on nectarines (Janisiewicz et al., 2013). Aggressiveness of Cercospora medicaginis or Phoma macdonaldii isolates was also correlated to a high growth and sporulation degree on media (Djebali et al., 2012; Roustaee et al., 2000). The length of the germ tube from the conidium to the first branch of the germ tube of M. fructicola isolates was greater than 100 μm when the fungi were grown on water agar plates (De Cal and Melgarejo, 1999) or in Czapek broth in the present study. The germ tube from the conidium to the first branch of the germ tube of M. laxa isolates was less than 60 μm when the fungi were grown on water agar plates (De Cal and Melgarejo, 1999). Batra (1979) also found that the growth rate of M. laxa is slower than that of M. fructigena and M. fructicola. M. fructigena grew 80 mm after 7 days of incubation, whereas M. laxa grew 40 mm after six days (Hrustić et al., 2012). In B. cinerea, pathogenicity was also dependent on growth characteristics of strains. Strains that produced abundant conidia (conidial morphological type) were on average more aggressive than strains that formed abundant sclerotia (sclerotial morphological type) (Korolev et al., 2008). Several factors observed in M. fructicola, such as greater growth associated with increased sporulation, greater aggressiveness on nectarines at postharvest and the presence of sclerotia that could increase their survival under unfavourable conditions, may explain the shift that M. fructicola is causing on the other Monilinia species in Ebro Valley (Spain). De Cal et al. (2013) reported previously that colonization of peaches and nectarines by M. fructicola was accompanied by local acidification of the host tissue, by gluconic acid accumulation that enhanced the expression of pectolytic enzymes that could facilitate pathogen development on fruit. Although macro-morphological characters are usually insufficient for an accurate M. fructicola identification (Lichtemberg et al., 2014), M. fructicola is the most distinct species of Monilinia among the three studied species as shown by the dendrograms generated from the presence of sclerotia and the lesion length on infected fruit. This dendrogram separated the 60 Monilinia isolates into two clusters, one cluster contained all the M. laxa and M. fructigena isolates and the second cluster comprising M. fructicola isolates. M. fructicola showed the highest aggressiveness components, followed by M. laxa, with M. fructigena in third place. Growth speed and sclerotia production on PDA and aggressiveness on detached leaves in vitro are indicators of Sclerotinia sclerotiorum and S. trifoliorum aggressiveness (Vleugels et al., 2013). The three Monilinia species were also separated in three clusters (r = 0.81) when each cluster comprised the isolates of each of the three Monilinia spp. (Fig. 3), when the presence of stromata and
Fig. 4. Sclerotia (a) and stroma (b) from Monilinia isolates on PDA colonies after 11 days of incubation at 22 ± 2 °C in the dark.
sclerotia on PDA (Fig. 4) and the lesion diameter on fruit were analysed. Two main types of stromata have been described in Sclerotinaceae, the sclerotial stroma and the “substratal stroma” (Byrde and Willetts, 1977), which are usually referred here to as the “sclerotia” and “stromata”, respectively. Both have been described as extreme important structures in the life cycles of those fungi that produce them. They serve as vegetative reproductive bodies; reproductive structures, asexual and sexual, may develop from them; and they are able to survive adverse conditions for long periods (Byrde and Willetts, 1977). In our investigation, we found that only the presence of stromata differentiates M. laxa from M. fructigena colonies when they were grown on PDA. Petróczy et al. (2012) reported no differences in the size, colour, and the presence of stromata in the colonies of M. fructigena and M. laxa when the two fungi were grown on PDA. Of the two species, M. laxa colonies displayed more stromata on culture. In fact, we found that stromata were always present in the colonies when our M. laxa isolates were cultured on PDA after 11 days of incubation. M. fructigena has always been reported to have slower stroma formation than in the other Monilinia species (Byrde and Willetts, 1977). Van Leeuwen et al. (2002) reported that the difference in spore size and the intensity of stromata formation on cherry agar medium are good discriminating cultural characteristics between M. polystroma and M. fructigena. In the last few years, M. polystroma has been reported from several European countries (Czech Republic, Switzerland, Poland, Serbia, Slovenia, and Italy) and from China, but not from Spain. The most significant symptoms on peaches infected by M. polystroma were a large number of yellowish or buff-coloured stromata and firm decayed tissues (Martini et al., 2014; Munda, 2015).
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In summary, we found that the studied M. fructicola isolates were the most aggressive on nectarine fruit at postharvest condition and showed the greatest growth differences from the other two species. Specifically, these isolates of M. fructicola differed greatly from M. laxa and M. fructigena by the presence of sclerotia when they were grown on PDA and the diameter of lesion they produced on fruit infected. The growth and aggressiveness components studied could explain, at least in part, the displacement of M. laxa and M. fructigena by M. fructicola recorded in Spanish peach orchards (Villarino et al., 2013), pointing to the importance of the fast growth rate and infection of M. fructicola and its sclerotia as the key component in dominating the other two species. Adaptive advantages of M. fructicola may lead to increased brown rot post-harvest losses. Acknowledgements This study was supported by grants AGL2011-30472-CO2 from the Ministry of Economy and Competition (Spain) and ERA37-DIMO (supported by INIA in EUPHRESCO Eranet). We thank the Postharvest Unit, Fruit Centre UdL-IRTA (Lleida, Spain) for their kind help and collaboration, R. Castillo and M.T. Morales-Clemente for their technical support. The authors would like to acknowledge Dr. Arieh Bomzon, Consul Write (www.consulwrite.com) for his editorial assistance in preparing this manuscript. References Badenes, M.L., Lorente, M., Martínez, J., Llácer, G., 1999. Variedades de Melocotón y Nectarina tempranas. Sèrie Divulgació Tècnica n° 46. Generalitat Valenciana: Consellería de Agricultura, Pesca y Alimentación. Batra, L.R., 1979. First authenticated North American record of Monilinia fructigena, with notes on related species. Mycotaxon 8, 476–484. Baxter, L.W., Zehr, E.I., Epps, W.M., 1974. A method for inducing production of apothecia of Monilinia fructicola from brown rot-infected stone fruit in South Carolina. Plant Dis. Rep. 58, 844–845. Biggs, A.R., Northover, J., 1985. Inoculum sources for Monilinia fructicola in Ontario peach orchards. Can. J. Plant Pathol. 7, 302–307. Boehm, E.W.A., Ma, Z., Michailides, T.J., 2001. Species-specific detection of Monilinia fructicola from California stone fruits and flowers. Phytopathology 91, 428–439. Byrde, R.J., Willetts, H.J., 1977. The Brown Rot Fungi of Fruit — Their Biology and Control. Pergamon Press, Oxford. De Cal, A., Melgarejo, P., 1999. Effects of long-wave UV light on Monilinia growth and identification of species. Plant Dis. 83, 62–65. De Cal, A., Gell, I., Usall, J., Viñas, I., Melgarejo, P., 2009. First report of brown rot caused by Monilinia fructicola in peach orchards in Ebro Valley, Spain. Plant Dis. 93, 763. De Cal, A., Sandín-España, P., Martinez, F., Egüen, B., Chien-Ming, C., Lee, M.H., Melgarejo, P., Prusky, D., 2013. Role of gluconic acid and pH modulation in virulence of Monilinia fructicola on peach fruit. Postharvest Biol. Technol. 86, 418–423. De Cal, A., Egüen, B., Melgarejo, P., 2014. Vegetative compatibility groups and sexual reproduction among Spanish Monilinia fructicola isolates obtained from peach and nectarine orchards, but not Monilinia laxa. Fungal Biol. 118, 484–494. Djebali, N., Gaamour, N., Badri1, M., Mrabet, M., Badri, Y., Elkahoui, S., Aouani, M.E., 2012. Phenotypic characterization of seven Cercospora medicaginis isolates infecting annual Medicago species in Tunisia. Afr. J. Microbiol. Res. 6 (11), 2761–2767. http://dx.doi. org/10.5897/AJMR11.1394. Gell, I., Cubero, J., Melgarejo, P., 2007. Two different PCR approaches for universal diagnosis of brown rot and identification of Monilinia spp. in stone fruit trees. J. Appl. Microbiol. 103, 2629–2637. Gell, I., De Cal, A., Torres, R., Usall, J., Melgarejo, P., 2009. Conidial density of Monilinia spp. on peach fruit surfaces in relation to the incidences of latent infections and brown rot. Eur. J. Plant Pathol. 123, 415–424. Gril, T., Celar, F., Munda, A., Javornik, B., Jakse, J., 2008. AFLP analysis of intraspecific variation between Monilinia laxa isolates from different hosts. Plant Dis. 92, 1616–1624. Guijarro, B., Melgarejo, P., De Cal, A., 2008. Influence of additives on adhesion of Penicillium frequentans conidia to peach fruit surfaces and relationship to the biocontrol of brown rot caused by Monilinia laxa. Int. J. Food Microbiol. 126, 24–29. Holtz, B.A., Michailides, T.J., Hong, C.X., 1998. Development of apothecia from stone fruit infected and stromatized by Monilinia fructicola in California. Plant Dis. 82, 1375–1380.
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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Growth and aggressiveness factors affecting Monilinia spp. survival peaches M. Villarino, P. Melgarejo, A. De Cal ⁎ Department of Plant Protection, INIA, Carretera de La Coruña km 7, 28040, Madrid, Spain
a r t i c l e
i n f o
Available online 15 February 2016 Keywords: M. laxa M. fructigena Occurrence of species Lesion lengthstroma Sclerotia
a b s t r a c t Brown rot of stone fruit is caused by three species of Monilinia, Monilinia laxa, M. fructigena, and M. fructicola. Eleven components of 20 different isolates of each of the three Monilinia species were analysed to determine distinct aggressiveness and growth characteristics among the three fungi. M. fructicola showed the greatest lesion diameter, and the lowest incubation and latency period on fruit postharvest, however isolates of M. fructigena exhibited less aggressiveness components. Five growth characteristics of M. fructicola could be used to distinguish M. fructicola from the other two species. The dendrogram generated from only the presence of sclerotia and lesion length on infected fruit separated the 60 isolates into two clusters (r = 0.93). One cluster was composed of the M. laxa and M. fructigena isolates and the other cluster comprised the M. fructicola isolates. However, the dendrogram generated based on the presence of stromata and sclerotia in the same colony of the three species when they were grown on potato dextrose agar, and the lesion diameter on fruit infected with each species separated the 60 isolates into three clusters (r = 0.81). Each cluster comprised the isolates of each of three Monilinia spp. We discussed the effect of M. fructicola growth and aggressiveness differences on the displacement of M. laxa and M. fructigena by M. fructicola recorded in Spanish peach orchards and their effect on brown rot at postharvest. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Brown rot caused by Monilinia laxa (Aderh et Rulh) Honey, M. fructicola (Wint.) Honey, and M. fructigena Honey in Whetzel (Byrde and Willetts, 1977), is an economically important disease of commercial stone fruit production and storage. These three brown rot fungi can infect both the fruit, causing substantial preharvest and postharvest losses, and the blossoms and twigs of stone fruit trees, causing blossom and twig blight (Byrde and Willetts, 1977). Postharvest losses are typically more severe than preharvest losses, and routinely occur during storage and transport, in some cases even affecting fruit at the processing stage (Hong et al., 1997). Until 2006, peach brown rot in Spain was caused either by M. laxa or M. fructigena only (De Cal and Melgarejo, 1999; Gell et al., 2009), with M. laxa the most prevalent (85–90%) (Larena et al., 2005). In 2006, M. fructicola was detected in peach orchards in the Ebro Valley, Lleida, Spain (De Cal et al., 2009). Since 2010, M. fructicola has displaced M. fructigena and co-existed with M. laxa in the similar relative frequency as M. laxa in three Ebro Valley peach orchards (Villarino et al., 2013). Six years after its first detection, the relative frequency of M. fructicola ⁎ Corresponding author at: Plant Protection Department, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Ctra. de la Coruña km 7, 28040, Madrid, Spain. E-mail address: [email protected] (A. De Cal).
http://dx.doi.org/10.1016/j.ijfoodmicro.2016.02.011 0168-1605/© 2016 Elsevier B.V. All rights reserved.
(absolute frequency normalized) increased on stored fruit, latent infections, blighted shoots, pruned branches, and mummified fruit on the trees (Villarino et al., 2013). However, the relative frequency of M. laxa in commercial orchards decreased progressively, especially on harvested fruit (Villarino et al., 2013). Co-existence between M. fructicola and M. laxa has been previously reported in Michigan and California (USA), where the two pathogens have somewhat different niches at early or late-season (Boehm et al., 2001), although M. fructicola is the most prevalent brown rot species in these states of USA. M. fructicola can be distinguished from M. laxa by growth characteristics such as colony shape, colour of its conidia, absence of hyphal anastomoses between germinating conidia, germ tube extension of the conidia before germ tube branching, and also by VCG (De Cal and Melgarejo, 1999; Mordue, 1979a, 1979b; Penrose et al., 1976). M. fructicola produces conidia and spermatia more abundantly than M. fructigena, and its conidia size and hyphal diameter are smaller than those of M. fructigena (Mordue, 1979a, 1979c). M. fructigena can be distinguished from M. laxa by its colony margins, the different conidial colour, and the length of conidial germ tubes (De Cal and Melgarejo, 1999; Mordue, 1979b, 1979c). Microconidia can be found on decaying fruit infected with M. fructicola and in cultures of M. laxa and M. fructicola which are older than 30 days (Ogawa et al., 1975). Irregular stromatal crusts and discoid sclerotia may develop on the agar surface or within the medium as colonies age. Of the three fungi, the apothecia of M. fructicola can be produced from pseudosclerotial mummified fruit
M. Villarino et al. / International Journal of Food Microbiology 224 (2016) 22–27
under field (Biggs and Northover, 1985; Byrde and Willetts, 1977; Holtz et al., 1998) or laboratory conditions (Baxter et al., 1974; De Cal et al., 2014; Holtz et al., 1998; Willetts and Harada, 1984). In contrast, the apothecia of M. fructigena and M. laxa are not found in the field (Janisiewicz et al., 2013; Villarino et al., 2010) and have not yet been produced under laboratory conditions (De Cal et al., 2014; Willetts and Harada, 1984). Infection efficiency, latent period, spore production rate, infection period, and lesion size were considered as components of the pathogen aggressiveness, although these characteristics could vary between isolates within a species (Pariaud et al., 2009). Infection and sporulation of Monilinia spp. could also be influenced by the saprophytic and pathogenic fitness of each species and climatic conditions. The main objective of this study was to compare the growth and aggressiveness factors of the three Monilinia spp. in culture media and on infected fruit at postharvest, in order to identify any features which could be related to their survival and to the displacement of M. laxa and M. fructigena by M. fructicola in Spanish peach orchards. 2. Materials and methods 2.1. Fungal isolates Sixty isolates (20 each of M. fructicola, M. fructigena, and M. laxa) from different stone fruit from across the world were studied. The host, geographical origin, year, and number of isolates are listed in Table 1. Isolation and culturing of the 60 isolates was done on potato dextrose agar (PDA) (Difco Laboratories) supplemented with 0.5 g streptomycin sulphate/l (Lab. Reig Jofré S.A., Barcelona, Spain). Forty-
Table 1 Geographical origin, host, year, and number of Monilinia isolates. Specie
Geographic origin
M. fructicola
USA Bellegarde, Balandrán, France Albesa, Lleida, Spain Alfarràs, Lleida, Spain Sudanell, Lleida, Spain Lleida, Spain Alfarràs, Lleida, Spain Lleida, Spain Hamilton, New Zealand M. fructigena Australia Japan Portugal A Coruña, Spain Jubia, A Coruña, Spain Jubia, A Coruña, Spain Lleida, Spain Bellegarde, Balandrán, France Lleida, Spain Albesa, Lleida, Spain Alfarràs, Lleida, Spain Sudanell, Lleida, Spain M. laxa South Africa Cehegin, Murcia, Spain Bellegarde, Balandrán, France Albesa, Lleida, Spain Alfarràs, Lleida, Spain Sudanell, Lleida, Spain Serós, Lleida, Spain Riba-roja d'Ebre, Tarragona, Spain Zaidi, Lleida, Spain Zaidi, Lleida, Spain Lleida, Spain Alfarràs, Lleida, Spain Gimenells, Lleida, Spain Sudanell, Lleida, Spain Riba-roja d'Ebre, Tarragona, Spain Albesa, Lleida, Spain
(–) No information.
Host
Isolation Number year of isolates
Plum Peach Peach Peach Nectarine Nectarine Peach Peach – Prunus spp. Apple Quince Plum Apple Plum Nectarine Peach Peach Peach Peach Nectarine Peach Apricot Peach Peach Peach Nectarine Nectarine Peach Peach Nectarine Peach Peach Peach Nectarine Peach Peach
1994 2001 2006 2006 2006 2006 2007 2007 2008 1971 1995 1996 1996 1996 1996 1998 2001 2006 2006 2006 2006 1996 1996 2001 2006 2006 2006 2006 2006 2006 2006 2007 2007 2007 2007 2007 2008
1 1 1 3 2 1 7 3 1 1 1 2 1 1 1 2 6 1 2 1 1 1 1 1 2 3 1 1 1 1 1 1 1 1 2 1 1
23
four isolates were collected from different cultivars of peaches and nectarines in the Ebro Valley, Spain. Sixteen isolates were sent from France, Portugal, New Zealand, Australia, Japan, and South Africa. The isolates were identified as M. fructicola, M. fructigena, or M. laxa, using their growth characteristics (De Cal and Melgarejo, 1999) and a PCR-based assay (Gell et al., 2007). In order to investigate the 60 isolates in the collection, each isolate was stored at − 80 °C in 20% glycerol (long-term storage) and at 4 °C on PDA slants in the dark (short-term storage). The isolates were grown on PDA at 22–25 °C in the dark for mycelium and spore production. 2.2. Aggressiveness components Aggressiveness of each isolate of all Monilinia spp. was tested on four nectarine fruit, cv. `Big Top´ which is an early variety with high productivity (Badenes et al., 1999). The nectarines were sanitized by immersing each fruit in a 1% NaOCl solution for 5 min, and then in 70% ethanol for 1 min. After two rinses in sterile distilled water (SDW), three puncture wounds were made in the skin of each fruit and 25 μl conidial suspension (104 conidia/ml SDW) were put on each wound. The inoculated fruits were then incubated at 22 ± 2 °C for 7 days under fluorescent lighting (100 μE/m2s with a 16-h photoperiod) in humidity chambers that were lined with moist paper (Guijarro et al., 2008). Each fruit was visually examined daily for symptoms of brown rot, and the incidence of brown rot caused by each Monilinia isolate was recorded (Guijarro et al., 2008). The percentage of fruit with brown rot symptoms (% brown rot incidence), the incubation period (the time interval between infection inoculation and the onset of symptom from that infection) and latency period (the time interval between infection inoculation and the onset of sporulation from that infection) (Pariaud et al., 2009) were recorded for each infected fruit. The daily lesion length (diameter in mm/day) was calculated from the individual measurements of the lesion's diameter on fruit that were made on each day of the 7-day incubation using regression analysis. To determine sporulation, the infected area was first excised from the fruit surface, and then suspended in 100 ml ethanol (70%), sonication for 5 min. This volume was filtered through glass wool, centrifuged at 14,040 g for 10 min at 4 °C (Sorvall RC 5C Plus, GMI) and the resultant pellet was transferred to 5 ml SDW. The number of conidia of Monilinia spp. on brown rot lesion was counted (conidia/cm2 of each infected area) using a hemacytometer and a light microscope. The complete experiment was repeated twice. 2.3. Growth components The six growth components were investigated for all 60 isolates. Two components, namely the percentage germination of conidia (%) and the length of the germ tubes (μm), were studied under light microscopy. Four components, namely colony growth rate (mm/day), in vitro sporulation (number of conidia/cm2), and the absence or presence of stromata, and sclerotia, were studied when the isolates were grown on PDA. 2.3.1. Germination components In order to produce conidia for studying the germination component, the isolates were grown on PDA or PDA that was amended with 1% acetone for seven days in the dark at 22 °C, as recommended by Pascual et al. (1990). Those colonies that did not produce any sporulation were maintained at 5–10 °C for 60 days, as recommended by Masri (1967) and Tamm and Flückiger (1993). The different ways of producing the spores could have influenced the results. Conidia were removed from the surface of the colony with a sterile scalpel, suspended in SDW, and then sonicated for one minute. A 15-μl droplet of the conidial suspension (106 conidia/ml) was spread on 30 μl of Czapek Broth (Difco) on sterilized slides, which were incubated for 24 h at 22 °C
24
M. Villarino et al. / International Journal of Food Microbiology 224 (2016) 22–27
Table 2 Comparison of the brown rot incidence, diameter of lesion, sporulation, and incubation and latency periods of Monilinia fructicola, M. fructigena, and M. laxa growing on nectarine fruit cv. `Big Top´. Species
Brown rot incidence (%)
Diameter of lesion (mm/day)
Sporulation (number of conidia/cm2)
Incubation period (days)
Latency period (days)
M. fructicola M. fructigena M. laxa MSE
88.1 a 72.2 b 82.1 a 399.8
3.6 a 2.2 c 3.0 b 1.9
0.7 × 106 b 1.1 × 106 b 3.3 × 106 a 1.8 × 1012
3.4 b 3.6 ab 3.7 a 0.2
4.0 b 4.4 a 4.5 a 0.2
Data are displayed as the mean of three wounds per fruit. Data were analysed by analysis of variance using the general linear model procedure in Statgraphics XVI Centurion for Windows 7 (StatPoint, Inc. Herndon, VA, USA). When the F test was significant at P = 0.05, the means were compared by the Student–Newman–Keuls multiple range test. Means followed by the same letter in each column are not significant at P = 0.05. MSE — mean squared error.
in the dark. All slides were dried under heat after incubation at the same time. Percentage germination was determined in 50 randomly selected conidia and germ tube length was measured in 25 randomly selected conidia using a light microscope (×100) (Zeiss Axioskop 2; Carl Zeiss, Inc.) with an ocular micrometre. Conidia were considered to have germinated when the germ tube was longer than the length of the conidia. Three replicates (drops) were made for each isolate and each assay was repeated twice. 2.3.2. Growth components on PDA The colony morphology of each isolate was determined on PDA. Plugs (ø 5 mm) of actively growing mycelia were cut from the margins of a 7-day-old colony of each isolate and placed in the centre of a sterile PDA Petri dish (ø 9 cm). The plates were then incubated for 11 days at 22 ± 2 °C in the dark. The absence or presence of stromata and sclerotia in the colony of each isolate was recorded visually on days 5 and 11 of the 11-day incubation. The daily growth rate (mm/day) was calculated from the individual measurements of colony diameter that were made on each day of the 11-day incubation using regression analysis. Sporulation was calculated after by suspending the entire colony of each isolate on PDA in 100 ml ethanol (70%), and sonicating for 5 min. The suspension was then filtered through glass wool and concentrated by centrifuging (Sorvall® RC 5C Plus, GMI) at 14,040 g for 10 min at 4 °C. The resultant pellet was transferred to 5 ml SDW, and the number of conidia of Monilinia spp. was counted using a cell counting chamber and a light microscope. Five replicate plates were used for each isolate and complete each assay was repeated twice. 2.4. Data analysis Data of in vitro and in vivo components of Monilinia were analysed by analysis of variance using the general linear model (GLM) procedure in Statgraphics XVI Centurion for Windows 7 (StatPoint, Inc. Herndon, VA,
USA). For this purpose, the following equation was used: Zijk ¼ μ þ Si þ IðSÞ jðiÞ þ ekðijÞ where μ is a “grand mean”, Z is the variable (in vitro and in vivo components of Monilinia), S (species) is the fixed factor, I (isolate) is nested to species, and e (error) is a random component due to factors that were not included in the model. When the F test was significant at P b 0.05, the means were compared by the Student–Newman–Keuls multiple range test (Snedecor and Cochram, 1980). Mean square error (MSE) and the marginal contribution of each main effect were considered, but only MSE was shown for measuring the contribution of each variable to the model when it is added in the order listed. The data were also analysed using a numerical taxonomy and multivariate analysis system NTSYS-pc version 2.10b (Exeter Software, Setauket, New York, USA) (Rohlf, 1993). Similarity matrices were constructed from the data using simple matching coefficient (SM) (Sokal and Michener, 1958). Dendrograms from the similarity matrices were generated using the unweighted pair-group method with arithmetic average (UPGMA) by the sequential, agglomerative, hierarchical, and nested (SAHN) clustering programme of NTSYS-pc version 2.10b. The goodness of fit of each cluster analysis was calculated by a cophenetic correlation coefficient (Rohlf and Sokal, 1981). When the value of a cophenetic correlation coefficient was ≥ 0.8, this value means that the data within a cluster are most likely to be highly reliable (Rohlf, 1993). The data of the 20 each M. fructicola isolates, M. fructigena, and M. laxa growing on PDA and on nectarine fruit cv. `Big Top´ were also subjected to principal component analysis (PCA) using the GenAlEx 6.2 software programme (Peakall and Smouse, 2006). 3. Results The incidence of brown rot caused by either the M. fructicola or M. laxa isolates was similar (up to 82%) and significantly higher than that of brown rot caused by M. fructigena isolates (72%) (Table 2). The fastest growth rate was in lesions caused by M. fructicola isolates (3.63 mm/day) and these lesions had the shortest incubation and latent periods (3.4 and 4.0 days, respectively) (Table 2). In contrast, sporulation on the infected fruit was greatest on those fruit inoculated with M. laxa (3 × 106 conidia/cm2). The length of the germ tubes was the greatest (51.56 μm) in M. fructicola isolates (Table 3). However, no differences on the germination percentage were observed among isolates. M. fructicola had the fastest colony growth rate (5.93 mm/day), the largest number of produced conidia (104 conidia/cm2), and the most sclerotia on PDA (Table 3). In contrast, M. laxa colonies showed the most stromata of all the colonies studied (Table 3). The results of the principal component analysis (PCA) revealed that the most important factor that distinguished species was the presence of sclerotia and stromata, followed, in declining order, by the lesion length on infected fruit, the germ tube length, the germination percentage, and the latency period (Fig. 1). Components 1, 2, 3, and 4 explained 29.31, 25.84, 17.94, and 15.93% of the variance, respectively.
Table 3 Comparison of the germination percentage, length of the germ tube, colony growth rate, sporulation, presence of sclerotia, and presence of stromata of Monilinia fructicola, M. fructigena, and M. laxa growing on potato dextrose agar (PDA). Species
Germination percentage (%)
Germ tube length (μm)
Colony growth rate (mm/day)
Presence of sclerotia
Presence of stromata
Sporulation (number of conidia/cm2)
M. fructicola M. fructigena M. laxa MSE
43.0 a 53.0 a 40.6 a 311.1
51.6 a 40.4 b 34.7 b 215.2
5.93 a 5.0 b 4.7 b 4.3
0.7 a 0.1 b 0.1 b 0.1
0.6 b 0.3 c 1.0 a 0.1
10,065.5 a 12.9 b 3.5 b 4.0 × 108
Data are displayed as the mean of four fruit with three replications. Data were analysed by analysis of variance using the general linear model procedure in Statgraphics XVI Centurion for Windows 7 (StatPoint, Inc. Herndon, VA, USA). When the F test was significant at P = 0.05, the means were compared by the Student–Newman–Keuls multiple range test. Means followed by the same letter in each column are not significant at P = 0.05. MSE — mean squared error.
M. Villarino et al. / International Journal of Food Microbiology 224 (2016) 22–27
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The dendrogram generated from the presence of sclerotia in the colonies of the 20 isolates each of M. fructicola, M. fructigena, and M. laxa isolates when they were grown on PDA and the lesion length on fruit infected with each 20 isolates of each M. fructicola, M. fructigena, and M. laxa separated the 60 isolates into two clusters (r = 0.93) (Fig. 2). The clustering of the isolates in the dendrogram was independent of the year of their isolation and the host. However, the dendrogram generated from the presence of stromata and sclerotia in the colonies of the 20 isolates of each M. fructicola, M. fructigena, and M. laxa when they were grown on PDA and the lesion length on fruit infected with each of the 20 isolates of each of M. fructicola, M. fructigena, and M. laxa separated the 60 isolates into three clusters (r = 0.81). Each cluster comprised the isolates of each of three Monilinia spp. (Fig. 3). Fig. 1. Principal component data analysis of the growth and aggressiveness factors from the 20 isolates each of Monilinia fructicola, M. fructigena, and M. laxa growing on potato dextrose agar (PDA) and on nectarine fruit cv. `Big Top´. PCA revealed the most important factor that distinguished species. Components 1, 2, 3 and 4 accounted for 29.31, 25.84, 17.94, and 15.93% of the variance, respectively.
4. Discussion All Monilinia isolates tested were pathogenic on nectarines at postharvest conditions. We found similar incidence of brown rot on infected
Fig. 2. Dendrogram generated from the presence of sclerotia in colonies of the 20 isolates each of Monilinia fructicola, M. fructigena, and M. laxa when they were grown on potato dextrose agar and the lesion length on fruit infected with each of the 60 isolates. The dendrogram was generated using the unweighted pair-group method with arithmetic average (UPGMA) by the sequential, agglomerative, hierarchical, and nested (SAHN) clustering programme of NTSYS-pc version 2.10b after calculating the simple matching (SM) coefficient. The cophenetic coefficient, r = 0.93.
Fig. 3. Dendrogram generated from the presence of sclerotia and stromata in the colonies of the 20 isolates each of Monilinia fructicola, M. fructigena, and M. laxa when they were grown on potato dextrose agar and lesion length on fruit infected with each of the 60 isolates. The dendrogram was generated using the unweighted pair-group method with arithmetic average (UPGMA) by the sequential, agglomerative, hierarchical, and nested (SAHN) clustering programme of NTSYS-pc version 2.10b after calculating the simple matching (SM) coefficient. The cophenetic coefficient, r = 0.81.
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fruit due to M. fructicola and M. laxa. However, isolates of M. fructicola developed the largest lesions, and the lowest incubation and latency period. Lesions, and the lowest incubation and latency period, have also been related to aggressiveness components (Pariaud et al., 2009). Isolates of M. fructigena resulted in less aggressiveness components. These findings indicate that M. fructigena was less aggressive on nectarine fruit than M. fructicola or M. laxa. Although the three pathogenic Monilinia spp. are polytrophs and can infect a wide range of Rosaceae spp., the pathogenic activity of M. fructigena as a cause of fruit rot is mainly confined to pome fruit (Gril et al., 2008). Of the three brown rot fungi, M. fructicola also exhibited the most differences in growth characteristics on media. Dispersal ability of M. fructicola could be considered better than either M. fructigena or M. laxa because it grows faster (Lichtemberg et al., 2014) and sporulates more abundantly (Byrde and Willetts, 1977). However, Hu et al. (2011) evaluated four Monilinia species for colony and lesion growth rates on PDA and peach fruit, and found that on PDA the fastest colony growth rate was observed for M. fructicola but M. laxa was faster in inoculated fruit. We also found five significantly different growth characteristics of M. fructicola from the other two species. M. fructicola showed the highest values in four of them with potential relationship to aggressiveness, length of germ tube, presence of sclerotia, growth rate, and sporulation. A significant relationship has been observed between growth and sporulation of M. fructicola isolates on PDA and their aggressiveness on nectarines (Janisiewicz et al., 2013). Aggressiveness of Cercospora medicaginis or Phoma macdonaldii isolates was also correlated to a high growth and sporulation degree on media (Djebali et al., 2012; Roustaee et al., 2000). The length of the germ tube from the conidium to the first branch of the germ tube of M. fructicola isolates was greater than 100 μm when the fungi were grown on water agar plates (De Cal and Melgarejo, 1999) or in Czapek broth in the present study. The germ tube from the conidium to the first branch of the germ tube of M. laxa isolates was less than 60 μm when the fungi were grown on water agar plates (De Cal and Melgarejo, 1999). Batra (1979) also found that the growth rate of M. laxa is slower than that of M. fructigena and M. fructicola. M. fructigena grew 80 mm after 7 days of incubation, whereas M. laxa grew 40 mm after six days (Hrustić et al., 2012). In B. cinerea, pathogenicity was also dependent on growth characteristics of strains. Strains that produced abundant conidia (conidial morphological type) were on average more aggressive than strains that formed abundant sclerotia (sclerotial morphological type) (Korolev et al., 2008). Several factors observed in M. fructicola, such as greater growth associated with increased sporulation, greater aggressiveness on nectarines at postharvest and the presence of sclerotia that could increase their survival under unfavourable conditions, may explain the shift that M. fructicola is causing on the other Monilinia species in Ebro Valley (Spain). De Cal et al. (2013) reported previously that colonization of peaches and nectarines by M. fructicola was accompanied by local acidification of the host tissue, by gluconic acid accumulation that enhanced the expression of pectolytic enzymes that could facilitate pathogen development on fruit. Although macro-morphological characters are usually insufficient for an accurate M. fructicola identification (Lichtemberg et al., 2014), M. fructicola is the most distinct species of Monilinia among the three studied species as shown by the dendrograms generated from the presence of sclerotia and the lesion length on infected fruit. This dendrogram separated the 60 Monilinia isolates into two clusters, one cluster contained all the M. laxa and M. fructigena isolates and the second cluster comprising M. fructicola isolates. M. fructicola showed the highest aggressiveness components, followed by M. laxa, with M. fructigena in third place. Growth speed and sclerotia production on PDA and aggressiveness on detached leaves in vitro are indicators of Sclerotinia sclerotiorum and S. trifoliorum aggressiveness (Vleugels et al., 2013). The three Monilinia species were also separated in three clusters (r = 0.81) when each cluster comprised the isolates of each of the three Monilinia spp. (Fig. 3), when the presence of stromata and
Fig. 4. Sclerotia (a) and stroma (b) from Monilinia isolates on PDA colonies after 11 days of incubation at 22 ± 2 °C in the dark.
sclerotia on PDA (Fig. 4) and the lesion diameter on fruit were analysed. Two main types of stromata have been described in Sclerotinaceae, the sclerotial stroma and the “substratal stroma” (Byrde and Willetts, 1977), which are usually referred here to as the “sclerotia” and “stromata”, respectively. Both have been described as extreme important structures in the life cycles of those fungi that produce them. They serve as vegetative reproductive bodies; reproductive structures, asexual and sexual, may develop from them; and they are able to survive adverse conditions for long periods (Byrde and Willetts, 1977). In our investigation, we found that only the presence of stromata differentiates M. laxa from M. fructigena colonies when they were grown on PDA. Petróczy et al. (2012) reported no differences in the size, colour, and the presence of stromata in the colonies of M. fructigena and M. laxa when the two fungi were grown on PDA. Of the two species, M. laxa colonies displayed more stromata on culture. In fact, we found that stromata were always present in the colonies when our M. laxa isolates were cultured on PDA after 11 days of incubation. M. fructigena has always been reported to have slower stroma formation than in the other Monilinia species (Byrde and Willetts, 1977). Van Leeuwen et al. (2002) reported that the difference in spore size and the intensity of stromata formation on cherry agar medium are good discriminating cultural characteristics between M. polystroma and M. fructigena. In the last few years, M. polystroma has been reported from several European countries (Czech Republic, Switzerland, Poland, Serbia, Slovenia, and Italy) and from China, but not from Spain. The most significant symptoms on peaches infected by M. polystroma were a large number of yellowish or buff-coloured stromata and firm decayed tissues (Martini et al., 2014; Munda, 2015).
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In summary, we found that the studied M. fructicola isolates were the most aggressive on nectarine fruit at postharvest condition and showed the greatest growth differences from the other two species. Specifically, these isolates of M. fructicola differed greatly from M. laxa and M. fructigena by the presence of sclerotia when they were grown on PDA and the diameter of lesion they produced on fruit infected. The growth and aggressiveness components studied could explain, at least in part, the displacement of M. laxa and M. fructigena by M. fructicola recorded in Spanish peach orchards (Villarino et al., 2013), pointing to the importance of the fast growth rate and infection of M. fructicola and its sclerotia as the key component in dominating the other two species. Adaptive advantages of M. fructicola may lead to increased brown rot post-harvest losses. Acknowledgements This study was supported by grants AGL2011-30472-CO2 from the Ministry of Economy and Competition (Spain) and ERA37-DIMO (supported by INIA in EUPHRESCO Eranet). We thank the Postharvest Unit, Fruit Centre UdL-IRTA (Lleida, Spain) for their kind help and collaboration, R. Castillo and M.T. Morales-Clemente for their technical support. The authors would like to acknowledge Dr. Arieh Bomzon, Consul Write (www.consulwrite.com) for his editorial assistance in preparing this manuscript. References Badenes, M.L., Lorente, M., Martínez, J., Llácer, G., 1999. Variedades de Melocotón y Nectarina tempranas. Sèrie Divulgació Tècnica n° 46. Generalitat Valenciana: Consellería de Agricultura, Pesca y Alimentación. Batra, L.R., 1979. First authenticated North American record of Monilinia fructigena, with notes on related species. Mycotaxon 8, 476–484. Baxter, L.W., Zehr, E.I., Epps, W.M., 1974. A method for inducing production of apothecia of Monilinia fructicola from brown rot-infected stone fruit in South Carolina. Plant Dis. Rep. 58, 844–845. Biggs, A.R., Northover, J., 1985. Inoculum sources for Monilinia fructicola in Ontario peach orchards. Can. J. Plant Pathol. 7, 302–307. Boehm, E.W.A., Ma, Z., Michailides, T.J., 2001. Species-specific detection of Monilinia fructicola from California stone fruits and flowers. Phytopathology 91, 428–439. Byrde, R.J., Willetts, H.J., 1977. The Brown Rot Fungi of Fruit — Their Biology and Control. Pergamon Press, Oxford. De Cal, A., Melgarejo, P., 1999. Effects of long-wave UV light on Monilinia growth and identification of species. Plant Dis. 83, 62–65. De Cal, A., Gell, I., Usall, J., Viñas, I., Melgarejo, P., 2009. First report of brown rot caused by Monilinia fructicola in peach orchards in Ebro Valley, Spain. Plant Dis. 93, 763. De Cal, A., Sandín-España, P., Martinez, F., Egüen, B., Chien-Ming, C., Lee, M.H., Melgarejo, P., Prusky, D., 2013. Role of gluconic acid and pH modulation in virulence of Monilinia fructicola on peach fruit. Postharvest Biol. Technol. 86, 418–423. De Cal, A., Egüen, B., Melgarejo, P., 2014. Vegetative compatibility groups and sexual reproduction among Spanish Monilinia fructicola isolates obtained from peach and nectarine orchards, but not Monilinia laxa. Fungal Biol. 118, 484–494. Djebali, N., Gaamour, N., Badri1, M., Mrabet, M., Badri, Y., Elkahoui, S., Aouani, M.E., 2012. Phenotypic characterization of seven Cercospora medicaginis isolates infecting annual Medicago species in Tunisia. Afr. J. Microbiol. Res. 6 (11), 2761–2767. http://dx.doi. org/10.5897/AJMR11.1394. Gell, I., Cubero, J., Melgarejo, P., 2007. Two different PCR approaches for universal diagnosis of brown rot and identification of Monilinia spp. in stone fruit trees. J. Appl. Microbiol. 103, 2629–2637. Gell, I., De Cal, A., Torres, R., Usall, J., Melgarejo, P., 2009. Conidial density of Monilinia spp. on peach fruit surfaces in relation to the incidences of latent infections and brown rot. Eur. J. Plant Pathol. 123, 415–424. Gril, T., Celar, F., Munda, A., Javornik, B., Jakse, J., 2008. AFLP analysis of intraspecific variation between Monilinia laxa isolates from different hosts. Plant Dis. 92, 1616–1624. Guijarro, B., Melgarejo, P., De Cal, A., 2008. Influence of additives on adhesion of Penicillium frequentans conidia to peach fruit surfaces and relationship to the biocontrol of brown rot caused by Monilinia laxa. Int. J. Food Microbiol. 126, 24–29. Holtz, B.A., Michailides, T.J., Hong, C.X., 1998. Development of apothecia from stone fruit infected and stromatized by Monilinia fructicola in California. Plant Dis. 82, 1375–1380.
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