Plant diseases need to be controlled in order to maintain global food availability, one of the three pillars of food security, and different approaches can be used to prevent, mitigate, or control plant diseases. In addition to good agronomic and horticultural practices, growers can apply chemical fertilizers and pesticides to their crops, and such applications have contributed significantly to spectacular improvements in food security. Finding non-chemical strategies for controlling crop diseases is of considerable interest because of environmental and health concerns about the widespread use of synthetic pesticides [1]. Nowadays, strict regulations on chemical pesticide use exist, and the political pressure to remove the most hazardous chemicals from the market is increasing. Therefore, the use of synthetic pesticides will probably be progressively reduced in the future, and this reduction will be accompanied by an increased reliance on the use of microorganisms as antagonists of plant pathogens [1]. However, controlling the wide spectrum of pathogens using microorganisms still remains an unfulfilled goal in commercial agriculture.

The main shortcoming of microbiological control is its lack of reliability, and several reasons can account for this limitation. Firstly, the population dynamics of the biological antagonist, the diversity of the natural population of the pathogen, and the natural climatic conditions are much more variable than those that are used in the laboratory to study the modes of actions of a biological control agent (BCA). Secondly, the climatic conditions that most favor the development of the pathogen might not be the same as the conditions that are required for the maximum expression of a BCA’s antagonistic activities [2]. Therefore, much more research should be devoted to identifying the environmental conditions that are required for the expression of a BCA’s beneficial effects under field conditions in order to make microbiological control more reliable.

Brown rot caused by Monilinia spp. is an economically important fungal disease of stone fruit, and is responsible for substantial pre- and postharvest losses [3]. Brown rot can be caused by the three Monilinia spp., namely M. laxa, M. fructicola, and M. fructigena [3]. Preharvest rains, the accumulation of excess moisture on fruit, wind, and the movement of insect vectors usually increase the incidence of brown rot [4]. Pre- and postharvest application of fungicides and insecticides to the fruit at proper intervals, the careful handling of harvested fruit in order to avoid wounding, proper pre- and postharvest sanitation, the rapid cooling of the harvested fruit and its storage at 0°C are all currently-used methods for decreasing brown rot incidence [5,6,7]. Despite the use of these disease control methods, brown rot at postharvest can still occur and cause significant crop losses [7,8,9]. Direct yield losses result from the infection of flowers (flower and twig blight), and from fruit rot at the preharvest, harvest, and postharvest stages [10]. Although the fruit can be infected with Monilinia spp. at any stage of its development, the disease only becomes severe when the fruit begins to ripen [11,12,13]. Susceptibility of peach fruit to M. laxa infection is usually greatest between the 32nd and 34th week of growth when the endocarp and pericarp are completely formed [13]. Postharvest losses are typically more severe than preharvest losses [10], and routinely occur during the fruit’s storage and transport [8]. Furthermore, development of pathogen resistance to synthetic pesticides [14,15,16,17,18], strong public opinion against the use of synthetic fungicides and insecticides, and the health risks that are associated with their application have made non-agrochemical products undesirable for controlling brown rot disease [19,20].

Epicoccum nigrum Link and Penicillium frequentans Westling are two common constituents of the resident mycobiota of peach twigs and flowers [21]. These two fungi have been shown to reduce twig blight caused by M. laxa in experimental stone fruit orchards [22,23,24] and postharvest brown rot caused by M. laxa and M. fructigena in laboratory assays [25,26,27]. Competition and antibiosis are the mode of action of these two fungal antagonists [24,28,29]. The major impediment in the commercialization of biocontrol products is the development of a formulation that will retain the same efficacy that is observed in laboratory assays in commercial orchards [30]. E. nigrum and P. frequentans have shown good potential for development as a commercial BCA against the brown rot of peach fruit [25,27,31]. Despite taking precautions to enhance BCA survival, severe environmental conditions may drastically limit a BCA’s ability to establish itself at its target site on the host [32]. Understanding the underlying ecological mechanisms of disease control and the population dynamics of a BCA in the environment is required when a BCA is proposed as a replacement for the existing methods of disease control [33]. Hence, the effect of inoculum type, the application frequency, and the time of application need to be zealously studied in order to ensure the efficacy of biological control [34,35].

Therefore, this article is an advanced survey of the nature and practice of biological control when it is used to control brown rot in stone fruit. This review will (i) describe the population dynamics of Penicillium frequentans and Epicoccum nigrum and their efficacy as BCAs against brown rot disease under field conditions, (ii) explore the relationships between antagonistic populations and biological control, and (iii) briefly outline future directions that might lead to the development of more diverse and effective BCAs.

P. frequentans and E. nigrum are effective biofungicides because they can reduce the number of Monilinia spp. conidia on fruit surfaces. The number of Monilinia spp. conidia on peach and nectarine fruit surfaces that were treated with either an E. nigrum or P. frequentans conidial formulation, as measured by the area under the conidial number of Monilinia spp. progress curve (AUncMOPC) are significantly lower (p= 0.05) than that found on untreated fruit surfaces (Table 1 and Table 2). A positive relationship between the conidial numbers of Monilinia spp. on peach surfaces and the incidence of latent infection has been reported [36], and these latent infections are also related to the incidence of postharvest brown rot [12]. The number of Monilinia spp. conidia on non-treated nectarine and peach surfaces increases from flowering to harvest. The numbers of Monilinia spp. conidia on fruit surfaces also increase rapidly during the pit hardening stage and during the month immediately before harvest, and the levels can be as high as 10^3.4–10^4.5 conidia per fruit surface. The highest levels of Monilinia spp. conidia on the fruit surface are usually found at harvest. The smallest Monilinia spp. conidial population was recorded on peach and nectarine fruit surfaces that were treated with a P. frequentans conidial formulation, followed by treatment with an E. nigrum conidial formulation and a fungicide (Table 1 and Table 2). In a previous study, we showed that an application of fresh E. nigrum or P. frequentans conidia at bloom and at preharvest reduced the incidence of postharvest brown rot in commercial peach orchards [31,37]. It has also been reported that preharvest application of other fungal BCAs, such as Metschnikowia fructicola, can also reduce the incidence of postharvest rot infections in stored strawberries [38]. Smilanick et al. [39] reported that preharvest infection of nectarines and peaches by Monilinia spp. could be the reason for poor control of brown rot when BCAs against this disease are applied postharvest.

One of the goals of a population dynamics study is to identify recurring patterns in the dynamics of the population, and to acquire knowledge on the mechanisms that generate these patterns [40]. The population dynamics of E. nigrum and P. frequentans in two commercial orchards in 2004 and 2005 are shown in Figure 1 and Figure 2, respectively. We found that the number of conidia (AUncPC) and the number of colony-forming units (CFUs) (AUcfuPC) for peach and nectarines trees that were treated with a P. frequentans conidial formulation were significantly higher (p = 0.05) than those for those trees that were treated with an E. nigrum conidial formulation (Table 1 and Table 2). In all instances, the number of E. nigrum and P. frequentans conidia on the peach surfaces (flowers or fruit) at each sampling date in every orchard was 100- to 1000-fold higher than the number of CFUs on Petri dishes that contained potato dextrose agar (PDA). Significantly larger E. nigrum and P. frequentans populations (p= 0.05) were present on the fruit surfaces 15 days before harvest (15–30% for the conidial populations and 80–100% for the number of CFUs) than on the flower surfaces in the early spring (Figure 1 and Figure 2). The number of E. nigrum and P. frequentans conidia remained fairly constant on the fruit surfaces over the period from petal fall to the first preharvest treatment with the conidial formulations (Figure 1 and Figure 2). Although the conidial concentration in the E. nigrum or P. frequentans formulations that were applied to the peach trees were the same, the size of the P. frequentans population on the surfaces was higher than that of the E. nigrum population.

Effective colonization, a large population size, and the viability of a BCA on plant surfaces are all essential for successful biocontrol of plant diseases [41]. We found that the numbers of E. nigrum and P. frequentans conidia are always significantly higher (p = 0.05) immediately following application of their conidial formulations when compared with those found at other sampling times (Figure 1 and Figure 2). It is possible that the greater number of P. frequentans conidia on the fruit surfaces may be due to a better adhesive ability than E. nigrum conidia, despite the ten-fold larger size of E. nigrum conidia [42]. In addition, the cell wall of E. nigrum conidia is composed of two layers whose outer layer is thick, studded with wart-like knobs, and pigmented by indole melanin [42].

The dynamics of individual populations within the epiphytic community are determined by the rates of immigration, emigration, growth, and death. Each of these factors is strongly influenced by the physical environment, chemical treatment, mean air temperature (tm), the relative humidity (RH), the amount of wind and rainfall, and solar radiation [40]. Hence, any climatic change, such as an increase in environmental temperature or drought stress, has the potential to affect a BCA’s performance and impact a food security agenda. Since fresh conidia without additives are more susceptible to environmental stress than dried conidial formulations, improving inoculum quality in such a manner as to reduce its susceptibility to environmental stresses is one way to increase the preharvest efficacy of BCAs [43]. We found that the sizes of the two BCA populations on the surface of peaches that were treated by either the FOR7 or FOR8 conidial formulation are much higher than those on the surfaces of peaches that were treated with fresh conidia without any additives [27,31]. We also found that the size of E. nigrum indigenous population was also lower than that of P. frequentans. Furthermore, application of both biological formulations always increased the size of the indigenous population of the fungi by 100- to 1000-fold on peach surfaces during the crop season in all tested orchards.

A principal component analysis (PCA) was undertaken to extract the linear composites of the number of Monilinia spp., E. nigrum and P. frequentans conidia on the nectarine and peach surfaces, the number of applications, the year of application, the formulation type, the tm, and the RH (Figure 3). According to the results of the PCA, we found that only the first three components had eigenvalues greater than one, and when combined, components 1, 2, and 3 accounted for 77.6% of the total variance, 41.3%, 23.5%, and 12.9%, respectively. The variance in the number of Monilinia spp. on fruit surface is mainly dependent on the number of E. nigrum and P. frequentans, the formulation type, and the RH (Figure 4). All were on the negative side of the first principal component axis, while the number of applications, the year of application, and the tm were on the positive side (Figure 4). The AUncMOPC and climatic conditions were on the negative side of the second principal component (Figure 4). On other hand, the AUncMOPC, the AUcfuPC, the RH, and the formulation type were closely grouped on the positive side of the third principal component axis (Figure 4). Shaw et al. [44] reported that the severity of a fungal disease in fruit is a function of temperature and wetness duration under controlled conditions. De Cal et al. [45] studied the population dynamics of E. nigrum on peaches and nectarines in order to find a method for promoting its colonization on fruit surfaces and improve its biocontrol efficacy against brown rot. They reported that the number of pit hardening and preharvest applications of the E. nigrum conidial formulations and RH accounted for 96% of the variability in the viability of the E. nigrum conidial population on the peach surfaces. In another study, Guijarro et al. [46] studied the population dynamics of P. frequentans on peach flower and fruit surfaces after different field treatments of six different P. frequentans conidia formulations. They found that size the P. frequentans conidial population on the surfaces was dependent upon application of the formulation to the blooming flowers and pre-harvested fruit, the RH, and the tm.

Preharvest application and optimization of BCAs require considerable understanding of the crop system, the pathogen’s epidemiology, and the biology, ecology, and population dynamics of the antagonists and their interactions [47]. The results of our studies suggest that the size of P. frequentans population after an application of a P. frequentans conidial formulation during the crop season is larger than that of E. nigrum following the application of an E. nigrum conidial formulation. Moreover, applications of a P. frequentans conidial formulation during the crop season also caused a greater reduction in the number of Monilinia spp. conidia on the fruit surface than that found after applications of an E. nigrum formulation during the growing season.

Biological control of brown rot in stone fruit caused by Monilinia spp. is very dependent on BCA density on the flower or fruit surface and the RH. One way to improve the performance of a fungal BCA is to develop an efficacious conidial formulation. The size of P. frequentans population after an application of a P. frequentans conidial formulation during the crop season is bigger than that of E. nigrum following the application of an E. nigrum conidial formulation. Moreover, applications of a P. frequentans conidial formulation during the crop season also caused a greater reduction in the number of Monilinia spp. conidia on the fruit surface than that found after applications of an E. nigrum formulation during the growing season. Finally, the potency of the E. nigrum conidial formulation is the same as that of fungicide.