Morphological, cultural and ecological characterization of Monilinia spp., pathogens of stone fruit in Serbia 632.4:579.24:579.6(497.11)Original

SUMMARY Different brown rot pathogens cause similar symptoms on fruit, which makes it difficult to differentiate them based only on developed symptoms. Therefore, several methods have been described for accurate identification of Monilinia species. In spite of the fact that those methods can be reliable, there are several limitations for their use, and the aim of this study was to find out if there are any specific characteristics that could be used as additional features for precise identification and in-depth characterization of Monilinia species. The results showed that morphological characteristics on PDA, and mycelium growth rate on PDA medium can be useful characters for differentiation of Monilinia species since M. fructicola was found to grow faster than the other two test species, while M. fructigena grew the slowest. MALT was the optimal medium for M. laxa and M. fructigena isolates, while PDA medium was optimal for M. fructicola . Regarding an extremely acidic medium (pH 2), only M. laxa and M. fructicola isolates were able to grow in it, while M. fructigena isolate was the only that grew in a moderately alkaline medium (pH 9). Also, the results revealed that the optimal temperature for M. fructigena and M. fructicola growth was 23ºC, while 28ºC was optimal for M. laxa isolate. On the other hand, some differences in the sensitivity of Monilinia isolates were revealed at extreme temperatures: M. fructigena isolate was the most sensitive (grew from 4°C to up to 31°C), while M. fructicola isolate was the most resistant (grew at 2°C and 34°C). The obtained results inferred that there are no specific features that can be used for reliable and precise identification of Monilinia species, but we observed some differences regarding the effects of extreme temperatures and pH values of culture medium on different species. Further research, involving more isolates, is needed for a final conclusion.


INTRODUCTION
Brown rot caused by Monilinia species is one of the most threatening diseases that cause important economic losses in pome and stone fruit production worldwide (Batra, 1991;Adaskaveg et al. 2008). Regarding stone fruit, three species of the Monilinia genus cause symptoms of brown rot: Monilinia laxa (Aderhold and Ruhland) Honey, Monilinia fructigena (Aderhold and Ruhland) Honey, and Monilinia fructicola (G. Winter) Honey (Byrde & Willetts, 1977;Batra, 1991). M. laxa is commonly found in all production areas worldwide, as well as in Serbia, while M. fructigena, regularly found in Europe, is a quarantine pathogen in the United States and Australia. On the other hand, M. fructicola is classified as a quarantine pathogen by the European and Mediterranean Plant Protection Organization (EPPO), and by the Serbian and Russian plant protection authorities, but it is widespread in stone fruit in the Americas and some parts of Africa and Asia. However, after its first introduction in France, M. fructicola became widespread in the vast majority of European countries, including Serbia. There is also the fourth species that might cause brown rot on stone fruit, Monilia polystroma, but this species is mainly detected on apple fruit (EFSA, 2011).
These brown rot pathogens cause similar symptoms on fruit, so that it is difficult to differentiate them based on symptoms alone. On the contrary, colony differences observed under laboratory conditions in vitro lead in most cases to accurate identification. Morphological identification, based on the key described by Lane (2002), is the simplest and the most convenient of recommended methods, confirmed in many studies (Fischer et al., 2017;Hrustić et al., 2012Hrustić et al., , 2015Lichtemberg et al., 2014;Papavasileiou et al., 2015;Poniatowska et al., 2013). However, morphological characteristics are easily affected by environmental conditions and cannot always be reliable for accurate species discrimination. Considering difficulties in performing accurate identification of Monilinia species on the basis of morphological characteristics, other methods have been developed. By far the most reliable results are provided by using molecular methods. The polymerase chain reaction (PCR) was used to develop many protocols for successful identification of Monilinia species. Some of these protocols, including the Multiplex protocol that allows differentiation of several Monilinia species in one PCR reaction (Côté et al., 2004), have been developed for distinguishing M. fructicola as the most destructive Monilinia species from other species of that genus (Hughes et al., 2000;Ioos & Frey, 2000). In spite of the fact that molecular methods are more reliable and accurate than morphological identification, there are several limitations to their use, such as their price per sample, and the availability of laboratory equipment and well-trained staff. Taking into account these obstacles, the aims of this study were: a) to find out if there are any specific cultural and/or ecological characteristics of Monilinia spp. that could be used as additional features for precise low-cost and reliable identification, and b) to provide more indepth cultural and ecological characterization of three Monilinia species.

Pathogen
From our previous four-year investigation (Hrustić et al., 2015) of 373 samples of stone fruit with brown rot symptoms that were collected and characterized, we selected three representative isolates of all three economically significant species, M. laxa, M. fructigena and M. fructicola, for further morphological, cultural and ecological characterization ( Table 1). The selected isolates were cultured on potato dextrose agar (PDA) medium at 20°C and stored at -80°C in 20% glycerol for long-term storage, and at 4°C on PDA slants for shortterm storage (Dhingra & Sinclair, 1995).

Morphological characteristics of isolates
Morphological characteristics of the isolates were investigated after 10-day incubation on PDA medium at 22 o C, as described by Lane (2002). The following parameters were observed: colony color, margin appearance, rosetting pattern, sporulation and presence of concentric rings of spores, medium pigmentation, and qualitative growth rate. Additionally, conidia produced on MALT medium after ten days incubation were observed and measured under light microscope at 100 × magnification (Olympus CX41, Japan). Also, the suspension of conidia was spread onto water agar (WA; 17 g agar, 1 l H 2 O) with sterile cotton swabs and incubated at 24°C in the dark for 18 h. After incubation, the percentage of germinated conidia of each isolate was calculated. A minimum of 50 conidia were examined for each isolate.

Mycelial growth rate assay
Three-millimeter diameter mycelial plugs were cut from the margin of seven-day old colonies, placed on PDA medium and incubated at the temperature of 24°C to asses mycelia growth rate of each tested isolate. Growth rate was calculated after seven days of incubation, and expressed as mm per day. The mean value of three replicates was used to represent each isolate.

Cultural characteristics of isolates
Mycelial growth was tested on five different media: PDA, V8 agar (200 ml V8 juice, 20 g agar, l l H 2 O), MALT (500 ml industrial malt, 17 g agar, 500 ml H 2 O), WA and CzA (3 g NaNO 3, 1 g K 2 HPO 4, 0.5 g MgSO 4 × 7H 2 O, 0.5 g KCl, 0.01 g FeSO 4 × 7H 2 O, 1 l H 2 O) as described above. Growth rate on each medium was calculated after seven days of incubation at 24°C in darkness, and expressed as mm per seven days. The mean value of three replicates was used to represent each isolate.
For evaluation of the effects of different pH values of the medium on colony growth rates of three Monilinia species, the pH of PDA medium was adjusted to range from 2 to 12 by using 0.1 N HCl or 0.1 N NaOH (Dhingra & Sinclair, 1995). Growth rate was calculated after seven days of incubation at 24°C in darkness, and expressed as mm per seven days. The mean value of three replicates was used to represent each isolate.

Ecological characteristics of isolates
The effect of various incubation temperatures on mycelial growth was evaluated on PDA medium. Threemillimeter diameter mycelial plugs were cut from the margin of seven days old colonies, placed on medium and incubated at 18, 20, 23, 25, 28 and 30°C. Additionally, effects of extreme temperatures were studied to determine the lowest and highest temperatures for mycelial growth per species. Temperatures lower than 4°C (0°C -4°C) and higher than 30°C (30°C -35°C) were tested. In all tests, colony diameter was measured in two perpendicular directions, after incubation of seven days. The trials were conducted independently in three replicates.

Statistical analysis
All data were analyzed by one-way analysis of variance (ANOVA) at 5% probability level with individual pairwise comparisons made by Tukey's test using the software Statistica 10 (2010) (Sokal & Rohlf, 1995).

Molecular identification of isolates
After morphological, cultural and ecological characterization, the identification of selected isolates was confirmed by polymerase chain reaction (PCR) using Multiplex PCR (Côté et al., 2004). Total amount of deoxyribonucleic acids (DNA) of the isolates was extracted from seven days old mycelia of the isolates grown on PDA medium with the DNeasy Plant Mini Kit (Qiagen, Germany) according to the manufacturer's instruction. The PCR reactions were performed in an Eppendorf Master Cycler (Eppendorf, Germany).

Morphological characteristics of isolates
The isolate of M. laxa had light-to dark-gray colony with lobed margins without sporulation, M. fructigena isolate had cream to yellow colony with rare sporulation and lobate margins, while the isolate of M. fructicola developed even margin hazel-colored zonate colonies with abundant sporulation and concentric rings of spores on the surface of PDA. According to described morphological characteristics determined by Lane (2002), the isolates were identified as M. laxa, M. fructigena and M. fructicola.
In general, the M. fructigena isolate produced the biggest conidia, 23.50 μm long and 13.50 μm wide,

Mycelial growth rate
At 24°C temperature, all three tested isolates showed statistically significant differences in colony growth rate (P<0.01). In general, the highest growth while M. laxa conidia were 15.42 μm long and 11.07 μm wide, and conidia of M. fructicola were 16.00 μm long and 10.75 μm wide. There was no statistical difference in the percentage of germinated conidia between the isolates of selected species (Table 2).  Growth rate (mm/7 days)

Cultural characteristics of isolates
Growth rates on five studied media showed that there were significant differences between the isolates of different species (P<0.05). All isolates were able to grow on all tested media, but generally the highest growth rate was recorded on MA and PDA media (Figure 1). Conversely, the lowest growth rate was recorded on the WA medium. Besides differences in growth rate, it was notice that all tested isolates had compact mycelium on PDA, MA and V8A media, while mycelium was transparent and sparse on WA and CzA media.
The isolate of M. laxa had the highest growth rate on MA medium (83.00 ± 1.79 mm/7 days) and the lowest on WA medium (31.67 ± 1.37 mm/7 days). Also, M. laxa produced conidia on MA medium after seven days of inoculation, while it occurred after 10 days of inoculation on PDA medium under daylight. Additionally, the isolate made characteristic rosette pattern colonies on PDA and MA media, while this characteristic pattern was not noticed on the other tested media.
M. fructigena had the highest growth rate (80.17±1.72 mm/7 days) on MA medium, and the lowest (15.33±7.15 mm/7 days) on CzA medium. Similar to M. laxa, the M. fructigena isolate produced conidia on PDA and MA media, while no conidia were produced on the other test media.
M. fructicola had the highest growth rate on PDA medium (71.33 ± 3.39 mm/7 days), and the lowest (17.67 ± 2.80 mm/7 days) on V8A medium. The isolate produced conidia abundantly on all tested media except WA medium, and concentric rings of conidia were especially evident on PDA medium.
In general, the optimal medium for M. laxa and M. fructigena isolates was MALT, whereas PDA was the optimal medium for M. fructicola.
The results of testing the effects of medium pH showed that there are statistically significant differences in growth rates on media from pH 3 to pH 9 (P<0.01). Only M. laxa and M. fructicola isolates were able to grow on extremely acidic medium (pH 2), and only M. fructigena isolate grew on moderately alkaline medium (pH 9) (Figure 2).
M. laxa had the highest growth rate on PDA medium pH 7 (73.75 ± 1.89 mm/7 days), and the isolate did not grow at pH 9 or higher. Additionally, the M. laxa isolate made a characteristic colony pattern on PDA medium from pH 5 to pH 8, which was not noticed on the other test media.
On the other hand, the M. fructigena isolate showed the highest growth rate on a medium with relatively low pH (pH 3, 30.50 ± 2.83 mm/7 days), and that was the only tested isolate that was able to grow at pH 10. Similar to M. laxa, the tested isolate of M. fructigena formed typical creamy to yellow colony with concentric rings of conidia, while its colonies were grey with rosette pattern at pH 3 and pH 4. The M. fructicola isolate had the highest growth rate on PDA medium of pH 6, and the lowest at pH 9, and there was no growth on pH exceeding 9. On all test media on which M. fructicola colonies were able to grow, they formed a typical colony pattern with concentric rings of conidia.

Ecological characteristics of isolates
The difference in growth rates of the isolates at different temperatures was statistically significant (P<0.01). The isolates of M. fructigena and M. fructicola had the highest growth rate at 23ºC, while the most favorable temperature for M. laxa was 28ºC ( Figure  3). On the other hand, the M. laxa and M. fructigena isolates were found to have the lowest growth rates at 18ºC, and the M. fructicola isolate at 28ºC. Besides differences in growth rates, the isolate of M. laxa also showed differences in morphological characteristics of colony shape as its colony had a typical rosette shape characteristic of M. laxa isolates at 23 and 25°C, but it had a rounded colony edge at other temperatures. On the other hand, temperature did not affect the colony shape of M. fructigena and M. fructicola isolates.
Aiming to assess the lowest and highest temperatures in terms of mycelial growth of each tested species, temperatures lower than 4°C and higher than 30°C were tested. The results showed that the M. fructigena isolate was the most sensitive to extreme temperatures: the lowest temperature at which M. fructigena colony grows was 4°C, and the highest was 31°C. On the other hand, the M. fructicola isolate was the most resistant: it grew at 2°C and 34°C. M. laxa showed moderate resistance to extreme temperatures: the lowest temperature at which its colony grew was 3°C, while it grew at temperatures lower than 33°C (Figure 4).

Molecular identification of isolates
Using the primers MO368-5, MO368-8R, MO368-10R and Laxa-R2, PCR products of predicted size were amplified: 352 bp for the M. laxa isolate, 402 bp for the M. fructigena isolate and 535 bp for the M. fructicola isolate, providing conformation of identification based on morphological, cultural and ecological characteristics. In the PCR test, no amplicon occurred in a negative control.

DISCUSSION
The results of our previous investigation showed that Monilinia spp. were dominant casual agents of brown rot of stone fruit in Serbia, which causes significant losses and damage (Hrustić et al., 2015;. In stone fruit production, more than one Monilinia species has been present in complex pathosystems, which makes control of these pathogens more challenging. Over the past eight years, three detailed information about their distribution, virulence, some morphological and molecular characteristics (Hrustić et al., 2015), as well as some information on the sensitivity of Monilinia spp. to most frequently used fungicides for brown rot control (Hrustić et al., 2018).
In the present study, we examined some specific cultural and/or ecological characteristics of Monilinia spp. that could be used as additional features for their precise, low-cost and reliable identification.
Morphological characteristics are widely used as identification criteria (Lane, 2002;Vasić et al., 2018;Fischer et al., 2017;De Cal & Melgarejo, 1999;van Leeuwen & van Kesteren, 1998). According to morphological characteristics described by Lane (2002), all isolates tested in this research had colonies with distinctive appearance: the M. laxa isolate had gray-colored colony with lobed margins typical for M. laxa, the M. fructigena isolate had yellow colony color with rare sporulation typical for M. fructigena, and the M. fructicola isolate had hazel-colored zonate colonies with abundant sporulation typical for M. fructicola. According to described characteristics and conformation of identification by using Multiplex PCR, the three chosen isolates were confirmed as M. laxa, M. fructigena and M. fructicola.
Besides morphological characteristics on PDA, mycelium growth rate on PDA medium can also be a useful character for differentiation of species of the Monilinia genus (Batra, 1979;van Leeuwen & van Kesteren, 1998;Lichtemberg et al., 2014;Poniatowska et al., 2013). Our results are consistent with some previous studies and showed that M. fructicola grew faster than the other two species and that M. fructigena grew the slowest.
Cultural and ecological features of different Monilinia species have not been widely described, and one of the main goals of this research was to provide an in-depth characterization of three economically important species of this genus. Our results revealed that the optimal medium for the tested M. laxa and M. fructigena isolates was MALT, whereas PDA was the optimal medium for M. fructicola. Similarly, Vasić et al. (2018) and Hu et al. (2011) showed that V8 and PDA media were the most favorable for the growth of Monilinia spp. Our results are also in agreement with findings reported by Vasić et al. (2018) that the optimal pH for M. laxa was 5-8, for M. fructigena 3-4, and for M. fructicola 6-8. Holb and Chauhan (2004) also reported that M. fructigena growth was faster at lower pH, between 2.5 and 6.5. Obi et al. (2018) reported the highest mycelial growth of a M. laxa isolate at pH 6.4, and the lowest at pH 2.4. Also, they proved that M. laxa is able to sporulate at a wide range of pH, between 3.5 and 9.5, while the optimum was found to be between pH 4.5 and 5.5.
The results of the present study revealed that the optimal temperature for M. fructigena and M. fructicola growth was 23ºC, and 28ºC for the M. laxa isolate. These results are partially in agreement with some previous studies (Vasić et al., 2018;Byrde & Willetts, 1977;Pereira et al., 2019;Papavasileiou et al., 2015). Fischer et al. (2017) reported that optimal growth rates from 20°C to 25°C, while Papavasileiou et al. (2015) found it to be at 25°C, and Lichtemberg et al. (2014) at 23.7°C. On the other hand, the ability to grow at extreme temperatures revealed some differences in the sensitivity of Monilinia isolates: M. fructigena was the most sensitive, while M. fructicola was the most resistant to extreme temperatures. Pereira et al. (2019) reported that minimum temperature averages for development of M. fructicola and M. laxa isolates were 4.5ºC and 4.0ºC, and the maximum temperature average for those isolates was 36ºC, while Papavasileiou et al. (2015) showed that 35ºC completely inhibited the growth of tested M. laxa and M. fructicola isolates from Greece.
Based on the results obtained in this investigation, we concluded that there are no specific cultural and/ or ecological features that can be utilized for reliable and precise identification of Monilinia species, and that only a comprehensive study including morphological, cultural, ecological and molecular characterization could lead to accurate determination of the causal agent of brown rot in stone fruit. Even though some differences in the effect of extreme temperatures and pH values of culture medium on different species was observed, further research involving more isolates is needed for a final conclusion. Also, our findings are consistent with a previous study (Villarino et al., 2016) in showing that the M. fructicola isolate had the greatest growth differences compared to the other two Monilinia species.

ACKNOWLEDGEMENT
This study was funded by the Ministry of Education, Science and Technological Development of the Republic of Serbia (Contract No. 451-03-68/2020-14/ 200214).