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The cape gooseberry (Physalis peruviana) crop in Colombia is important due to its economic impact, given the growing demand for this fruit in international markets (ICA, 2022). In the first seven months of 2022, fresh exports increased by 4.1% in comparison with the same period in 2021, with a FOB value of $24.6 million, USD (ANALDEX, 2022), adding 5.29 t, equivalent to an increase of 10.4% in comparison with the same period in 2021 (ANALDEX, 2022). The total production in 2021 was 19.37 t in 1.44 ha, with an average yield of 11 t ha-1, mainly in the departments of Cundinamarca (419 ha), Boyacá (404 ha), Nariño (204 ha) and Antioquia (146 ha), which represented 84% of the national production (AGRONET, 2022).
The crop also has a meaningful social impact due to the significant contribution towards food security, since in production costs, including harvest and postharvest, 45% corresponds to female workforce, heads of family. On the other hand, the productive chain demands additional workforce, since the cape gooseberry that is not exported (which can be 40%) is transformed into dried fruit, jams, sauces, syrups, and other subproducts that serve the national market (ICA, 2022).
The cape gooseberry is a fleshy and juicy yellow-orange berry known for its organoleptic properties (flavor, odor, and color) and its nutritional value since it contains vitamins A, B, and C, Fe and P, fiber, carotenoids, and flavonoids, known for their antioxidant properties (Puente et al., 2011). The fruit is formed from solitary hermaphroditic flowers, with an accrescent calyx that, in the maturity of the fruit, reaches a length of 4 to 5 cm, known in Colombia as the capacho, which encloses and protects the fruit from several environmental conditions (pests, rain and cold) (Fischer and Lüdders, 1997; Nocetti et al., 2020). Nevertheless, the calyx is susceptible to infection from several phytopathogens, such as Cladosporium sp., Cercospora sp., Sclerotinia sp., and Botrytis cinerea, the latter being the main limitation in production and postharvest (Forero, 2014). B. cinerea is the causal agent of gray mold, a cosmopolitan fungus that appears naturally in crops, affecting more than 250 plant species of agricultural interest. It is considered the second most limiting phytopathogen in agricultural production in the world, given its ability to environmental adaptation in the world, the costs related to its management, and the speed with which it can develop resistance to the fungicides used for their control (Dean et al., 2012; Hahn, 2014; Carisse, 2016; Wenyong et al., 2021).
Under field conditions, B. cinerea infects the calyx, causing disease if the environmental conditions are optimal (relative humidity ≥ 80% and temperatures between 12 and 22 °C). However, if they are not favorable, the pathogen remains quiescent. It aggressively reactivates the infection when favorable conditions coincide with calyx´s senescence and the fruit´s maturation, particularly during the transportation in its export (which may last between 15 and 20 days). Most fruits may be infected yet are asymptomatic when harvested, but the expression of the disease is shown when the fruit arrives at its destination, causing losses of more than 15% (Molina et al., 2004; Prusky and Lichter,2007; Prusky et al., 2013; Forero, 2014).
The gray mold is usually controlled with chemical fungicides. However, the resistance, food safety, harmful effects on the environment and human health, as well as the sustainability of the crop, increasingly limit the use of these chemical products, since in order for the fruits to be exported, mainly to Europe, compliance with the GLOBAL G.A.P standard is required, which restricts the residual concentration of different active ingredients of pesticides to minimum levels (Rincón et al., 2015). The above produces the need to evaluate and select environmentally friendly management alternatives such as biopesticides, plant extracts, or biological inoculations, which are allowed and promoted in different production systems in the United States and the European Union since they generally pose little to no threat to the environment and humans (Bautista et al., 2018). Their use in the cape gooseberry crop helps reduce the quiescent B. cinerea infections and reduces losses produced during the postharvest of the fruit. The aim of this work was to determine the efficacy of the application in the field of biopesticides based on Trichoderma koningiopsis Th003 and Rhodotorula mucilaginosa Lv316, a biological inoculant based on Bacillus amyloliquefaciens Bs006, on a biostimulant (Kendal®) and an extract of Swinglea glutinosa (EcoSwing®) on the reduction of the quiescent B. cinerea infections, considering previous studies carried out on berry crops, in which these treatments displayed and efficacy in the control of the fungus of up to 60% (Zapata and Cotes, 2013, Hincapié et al., 2017; Zapata and Beltrán, 2019).
Materials and methods
Incidence of gray mold on fruits. From four commercial crops, one in the municipal area of Ubaté (locality La Patera), another in the municipal area of Sutatausa (locality Hato Viejo), and two in the municipal area of Granada (locality La Veintidós sector Alto and La Veintidós sector bajo), 100 asymptomatic cape gooseberry fruit samples were gathered, packed in paper bags, and transported in styrofoam coolers for their analysis in the AGROSAVIA Agricultural Microbiology Lab. The fruits were placed in wet chambers with a relative humidity of ≥ 90% (HR) in sealed plastic containers measuring 14.5 x 24 x 36.5 cm (each one on a 1.70-ounce plastic cup) and stored in a room at 22 °C for seven days and next, the incidence of gray mold was measured according to the characteristic signs of the disease.
Evaluation of alternatives for management in the field. The experiment was established on the field with 220 cape gooseberry plants of the variety Corpoica-Dorada (Sánchez et al., 2016) in the locality La Veintidós sector alto of the municipal area of Granada, department of Cundinamarca, Colombia (1.800 masl). During the seedling stage, the cape gooseberry plants were inoculated with B. amyloliquefaciens Bs006 (the active component of the bioproduct Natibac® SC) by spraying the substrate at the moment of planting, as well as 7 and 21 days after planting. On the field, the bacteria were applied as a drench at the moment of transplanting, as well as 7 and 15 days after this process, at a concentration of 1x108 UFC mL-1, as a strategy to promote plant growth and increase their tolerance to biotic and abiotic stress (Beltrán-Acosta et al., 2023). Chemical fertilization was carried out following a scheme established according to a chemical soil analysis. The postharvest analyses of the fruits were carried out in the Agricultural Microbiology Lab of the AGROSAVIA Tibaitatá Research Center.
When the cape gooseberry fruits began to form (approximately six months after planting), foliar applications of T. koningiopsis Th003 (Tricotec® WG), of the yeast R. mucilaginosa Lv316 (Nalev® WG) and B. amyloliquefaciens Bs006 (Natibac® SC) were carried out, as well as a treatment called Eco, consisting of three applications of a biostimulant containing oligosaccharides and glutathione (Kendal®) and three applications of S. glutinosa (EcoSwing®) extract was applied, as well as a chemical treatment that consisted of alternate applications, three of a fungicide based on Azoxystrobin - Difenoconazole and three of a fungicide based on Thiram - pyrimethanil (Table 1).
Table 1 Treatments evaluated for the control of gray mold (Botrytis cinerea) in the cape gooseberry crop.
| Tratamiento | Dosis y concentración de aplicación |
|---|---|
| T. koningiopsis Th003 (Tricotec® WG) | 1 g L-1 / 1x106 conidios mL-1 |
| R. mucilaginosa Lv316 (Nalev® WG) | 2 g L-1 / 1x107 células mL-1 |
| B. amyloliquefaciens Bs006 (Natibac®SC) | 50 mL L-1 / 1x108 UFC mL-1 |
| Eco (Kendal® / EcoSwing®) | 1 mL L-1 / 1.5 mL L-1 |
| Químico (Azoxistrobina - Difenoconazole / Thiram - Pyrimethanil) | 1.5 mL L-1 / 2 mL L-1 |
The experiment was established under a randomized complete block design with three repetitions, where the experimental unit (EU) consisted of two rows with five plants each (10 plants per EU) and 30 plants per treatment. A row of untreated plants separated each treatment. As a control, fruits were taken from a crop planted simultaneously in the same plot, but in a different area, with the same variety and fertilization scheme, without using fungicides to control of foliar diseases. The variable evaluated was the incidence of gray mold.
The applications were carried out in the morning using a backpack sprayer with a total of five applications at a frequency of 15 days. After the third application, 30 ripe fruits were taken every week from each repetition per treatment; they were packed in paper bags and transported in styrofoam coolers for their analysis in the lab. Once there, 18 fruits with calyxes were taken from each repetition and placed in a wet chamber in the conditions described earlier for seven days. In addition, 15 fruits were taken from the plants, their calyxes were removed, and they were placed under the same conditions.
Follow-up of the populations of antagonists in the phyllosphere. After the first application of the treatments and before the following applications, ten leaves were taken randomly from each repetition (30 leaves per treatment). The leaves were stored in paper bags, labeled, and transported in a styrofoam cooler for their analysis in the lab. Once there, the leaves from each repetition were cut into pieces measuring 1 cm in diameter using a sterile stainless steel hole puncher, 10 g were taken from the plant material and placed in an Erlenmeyer with 90 mL of Tween 80 at 0.1% (mother suspension) leaving them to be stirred constantly at 150 rpm for one hour. Next, a 1 in 10 dilution was performed for each one, they were homogenized in a vortex stirrer, and 100 µL were taken and placed in Petri dishes (three replicas per dilution) containing Rose Bengal Agar for T. koningiopsis Th003, malt extract agar for R. mucilaginosa Lv316 and Luria Bertani agar for B. amyloliquefaciens Bs006, respectively. The aliquot was homogenously distributed with a stainless steel Drigalsky spatula. The dishes were incubated at 28 °C for 48 for the bacteria and 25 °C for the yeast and fungus for 48 hours and 5 days, respectively. After this time, the culture forming units (CFUs) were counted, and the result was reported as CFU/g of leaflet [expressed as Log (UFC/g)].
Data analysis. For every treatment, the healthy fruits were added up along with those that displayed signs and symptoms of gray mold, and the percentage of incidence was determined using the following formula: Percentage of incidence = (Fruits with gray mold /Total fruits) *100. The data were analyzed with an analysis of variance and a means comparison using Fisher’s LSD test (*= P>0.05) using the statistical software Statistix® 10.0. The efficacy in the control of the disease was estimated using Abbott’s formula: Percentage of efficacy = ((Cd - Td) / Cd)) *100. Where: Cd = Incidence in the control treatment and Td= Incidence per treatment (ANDI y ICA, 2015).
Results and discussion
Incidence of the gray mold on fruits from quiescent B. cinerea infections in commercial crops. The calyx of the cape gooseberry encloses and protects the fruit from the environment (Fischer and Lüdders, 1997; Nocetti et al., 2020). However, this exposure makes it susceptible to quiescent B. cinerea infections, and its senescence during the maturation of the fruit and a favorable environment during postharvest activate these infections and develop the disease (Figure 1). This condition was observed in this study, where the incidence of the gray mold from the quiescent B. cinerea infections in the fruits gathered surpassed 88% and even reached 100% in the crop of the locality La Veintidós sector bajo (Figures 2 and 3), showing that 80% of the fruit harvested and with an export potential can be infected with B. cinerea.
Figure 1 Top, healthy cape gooseberry (Physalis peruviana) fruits. Below, fruits with signs of gray mold (Botrytis cinerea) in the field, showing the presence of the mycelia and conidia of Botrytis cinerea with a grayish tome on the calyx and the berry inside.
Despite the lack of epidemiological studies that help establish an infection model for B. cinerea in cape gooseberry, the importance of quiescent infections has been proven in the losses the disease can cause in postharvest in other production systems in which the gray mold is one of the main limitations in their production. For example, investigations carried out on berries (Rubus sp.) have shown that this pathogen produces quiescent infections in all the physiological stages of the development of the fruit, leading to an incidence of gray mold in mature fruits of 60 to 80% (Molina et al., 2004), in the same way as Petrasch et al. (2019) and Rivera et al. (2013) have described for strawberries (Fragaria sp.) and cranberries (Vaccinium corymbosum), respectively.
Evaluation of alternatives for control in the field. The highest gray mold incidence was observed for the first two crops, with values between 60 and 84% (Figure 4). This period was characterized by precipitations with an average of 194 mm/day, which could favor the incidence of the disease. However, once this weather condition passed, and except for the control, which displayed a mean incidence of 83% throughout the evaluation period, the incidence in the treatments decreased, with the applications of Tricotec® WG and Nalev® WG standing out due to their mean incidences of 48 and 51%, respectively.
Figure 2 Incidence of gray mold (Botrytis cinerea) in cape gooseberry harvested in crops found in three municipal areas of Cundinamarca. Columns with the same letter are not significantly different, according to Fisher’s LSD test (*= P>0.05).
Figure 3 Cape gooseberry fruits with signs of gray mold (Botrytis cinerea) after their storage in wet chambers.
Nevertheless, the chemi cal treatment that displayed a mean incidence of 55% displayed the lowest incidence towards the end of the evaluation period, with 10% (Figure 4), coinciding with the applications of the Thiram - Pyrimethanil-based fungicide, a botrycide that in Colombia is not frequently used in cape gooseberry (unlike Azoxystrobin - Difenoconazole), although it is used in rose (Rosa sp.) crops. It is probable that due to the low exposure to the fungicide, the populations of B. cinerea in the cape gooseberry crop did not yet show resistance; however, in order to confirm this, studies are required to help determine the resistance of B. cinerea populations in crops in different locations to this and other fungicides.
Figure 4 Weekly gray mold (Botrytis cinerea) incidence in the fruit harvested by treatment. Lines with the same line are not significantly different, according to Fisher’s LSD test (*= P>0.05).
The efficacy of the control of the gray mold was established, depending on the reduction of the incidence, noticing that all treatments presented some level of control; however, with the applications of T. koningiopsis Th003 and R. mucilaginosa Lv316, efficacies of 42 and 39% were obtained, respectively, making these the most prominent treatments (Figure 5).
Figure 5 Efficacy of the treatments in the control of gray mold (Botrytis cinerea) in cape gooseberry fruits after 75 days of evaluation in the field.
Similar results in the control of B. cinerea have been reported for the berry crop, in which the application of the same biopesticides under a similar application scheme gave a control efficacy of 60%, higher than that obtained with the applications of Procloraz (58%) or Carbendazim (27%) (Zapata and Cotes, 2013). Regarding the control of B. cinerea with T. koningiopsis and R. mucilaginosa, it is necessary to consider that this pathogen is susceptible to the lack of nutrients since it limits the germination of conidia, the formation of the germinal tube and infections (Elad,1996). Therefore, the application of these antagonists, with action modes such as the competition for space and nutrients, mycoparasitism, and antibiosis, was able to reduce the infection of the pathogen in the calyx (Freimoser et al., 2019; Moreno-Velandia et al., 2020).
On the other hand, the application of the Kendal® rotation and the extract of S. glutinosa with an efficiency of 34% (Figure 5) proves to be an alternative worth considering in the management of the disease. An example of this was the 65% reduction of the incidence of gray mold obtained with its applications alternated with Tricotec® WG in the berry crop (Zapata and Beltrán, 2019) or in the control of downy mildew in berries in rotation with biopesticides based on Trichoderma harzianum and Bacillus subtilis, an extract of citrus seeds and copper-based fungicides (Boyzo-Marín et al., 2015).
The oligosaccharins and glutathione found in Kendal® are considered biostimulating molecules that act as elicitors associated with the stimulation of defense response in plants (Guevara et al., 2010; Garcia-Brugger et al., 2006), whereas the S. glutinosa extract contains α-pinene and β-pinene, compounds with inhibiting action on the germination of conidia and mycelial growth (Camargo-Piñeres et al., 2021). The combination of these mechanisms assists in the control of the pathogen in the plants treated.
On the other hand, the applications of the fungicides (chemical treatment) gave an efficacy of 29% (Figure 5), and considering that a lower incidence was observed at the end of the biotest with the applications of Thiram - Pyrimethanil, a rotation of products must be integrated into future studies in order to implement management strategies for the gray mold. It is convenient to evaluate the integration of the alternatives evaluated in this paper, applying them according to the weather conditions to obtain a better result and contribute to reducing the risk of fungicide-resistant B. cinerea populations.
In contrast, when removing the calyx from the fruit before storing, the incidence of gray mold did not surpass 2% (Figure 6), which leads us to assume that the quiescent B. cinerea infection is mainly produced in the calyx and from there, the pathogen is passed on to the fruit, or a microclimate is produced that is favorable for the fungus; unlike the infections in berries and strawberries, where the quiescent infections are produced in floral structures such as the stamen, carpel or the floral receptacle (Molina et al., 2004; Petrasch et al., 2019). According to this, the calyx could be removed from the fruit to reduce the risk of quiescent infections, although one of the market conditions, particularly in Europe, is the presence of the calyx on the fruit, making this alternative unviable.
Figure 6 Accumulated incidence after 75 days of evaluation for gray mold (Botrytis cinerea) by treatment in cape gooseberry fruits without a calyx. Columns with the same letter are not significantly different, according to Fisher’s LSD test (*= P>0.05).
Follow-up of the populations of antagonists in the phyllosphere. The phyllosphere is a hostile habitat for microorganisms: the low availability of nutrients, extreme temperatures, and solar radiation intensity make establishing antagonists difficult. Due to this, an inherent condition in an antagonist applied to the phyllosphere is its ability to adapt, colonize and remain in it since; otherwise, it will not carry out the control activity it was chosen to do (Andrews, 1992; Andrews and Harris, 2000). This study noticed that the antagonists colonized the cape gooseberry phyllosphere since their populations remained constant during the evaluation period, obtaining 1x103 UFC g-1 for T. koningiopsis Th003 per sampling, while R. mucilaginosa Lv316 y B. amyloliquefaciens Bs006 were obtained 1x105 UFC g-1 (Figure 7).
Figure 7 Follow-up of the population of antagonists applied on cape gooseberry plants during the evaluation period (October 7 to December 12, 2022).
Similar reports have been provided by authors such as Sylla and collaborators (2013), who applied biopesticides based on Trichoderma harzianum and B. amyloliquefaciens weekly for the control of B. cinerea in strawberries and obtained recounts of the phyllosphere of 1x102 UFC g-1 for the fungus and 2x104 UFC g-1 for the bacteria. Meanwhile, Elad and collaborators (1994) applied the yeasts Rhodotorula glutinis and Cryptococcus albidus for the control of B. cinerea in tomato and recovered, from the phyllosphere, populations of 8x103 UFC cm-2.
Conclusions
Applying biopesticides based on T. koningiopsis Th003 and R. mucilaginosa Lv316 on the field gave an efficacy in the reduction of quiescent B. cinerea infections of 42 and 39% respectively, in comparison with the 29% obtained with the alternated applications of Azoxistrobina - Difenoconazole and Thiram - Pyrimethanil-based fungicides. These results pose the integration of biopesticides as viable and efficient in the integrated management strategies for the crop, thus contributing to reducing the number of fungicide applications in the field.
