Postharvest biological control of brown rot in peaches after cold storage preceded by preharvest chemical control 1

Controle biológico pós-colheita da podridão parda em pêssego, após armazenamento refrigerado, antecedido de controle químico pré-colheita

ABSTRACT

Pathogenic fungi cause skin darkening and peach quality depreciation in post harvest. Therefore, alternative techniques to chemical treatment are necessary in order to reduce risks to human health. The aim of this study was to evaluate the effect of the application of Trichoderma harzianum in association with different fungicides applied before harvest to 'Eldorado' peaches for brown rot control and other quality parameters during storage. The treatments consisted of five preharvest fungicide applications (control, captan, iprodione, iminoctadine and tebuconazole) associated with postharvest application of T. harzianum, after cold storage (with and without application), in three evaluation times (zero, two and four days at 20 °C), resulting in a 5x2x3 factorial design. The application of T. harzianum only brought benefits to the control of brown rot when combined with the fungicide captan, at zero day shelf life. After two days, there was a greater skin darkening in peaches treated with T. harzianum compared with peaches without the treatment, except for peaches treated with the fungicide iprodione and T. harzianum The application of T. harzianum during postharvest showed no benefits for the control of brown rot, however, the association with fungicides reduced the incidence of Rhizopus stolonifer during the shelf life.

Key words:
Monilinia fructicola; Trichoderma harzianu;, quality; skin browning

RESUMO

Fungos fitopatogênicos causam escurecimento da epiderme e depreciação da qualidade de pêssegos em pós-colheita. Dessa forma, é preciso técnicas alternativas ao tratamento químico visando a reduzir os riscos para a saúde humana. O objetivo deste trabalho foi avaliar o efeito da aplicação de Trichoderma harzianum e sua associação com diferentes fungicidas aplicados pré-colheita em pêssegos 'Eldorado' para o controle da podridão parda e manutenção dos parâmetros de qualidade durante o armazenamento. Os tratamentos foram compostos por cinco aplicações pré-colheita de fungicidas (testemunha, captana, iprodiona, iminoctadina e tebuconazol) associado à aplicação pós-colheita de T.harzianum, após o armazenamento refrigerado (com e sem aplicação), mais três épocas de avaliações (zero, dois e quatro dias a 20 ºC), resultando em um esquema fatorial (5x2x3). A aplicação de T. harzianum só trouxe benefícios no controle de podridão parda quando associado ao fungicida captam a zero dias de vida de prateleira. Exceto quando os pêssegos foram submetidos à iprodione e T. harzianum, observou-se, após dois dias, maior escurecimento da epiderme em pêssegos com T. harzianum em comparação com pêssegos sem aplicação. A aplicação de T. harzianum durante a pós-colheita não mostrou benefícios no controle da podridão parda, no entanto, a associação com fungicidas, reduz a incidência de Rhizopus stolonifer durante a vida de prateleira.

Palavras-chave:
Monilinia fructicola; Trichoderma harzianum; qualidade; escurecimento da epiderme

Introduction

In the year 2012, Brazil ranked 14^th in the global production of peaches, producing 220.000 tonnes, which correspond to 1% of the world's total production (FAO, 2012). In Brazil, the state of Rio Grande do Sul is the leading producer of peaches, with 86% of the total cultivated area (Agrianual, 2011). Nevertheless, some problems decrease the fruit quality during the development and postharvest life of peach, especially brown rot incidence.

Brown rot is caused by the fungus Monilinia fructicola (G. Wint.) Honey and is one of the most important diseases affecting peaches. The most typical symptoms observed in peach flowers are the necrosis of anthers, peduncle and ovary (May-De Mio et al., 2004). In the fruits, small brown lesions are observed at first, which later change into extended brown lesions. When the infection is severe, an intervention has to be made to control this disease.

Chemical control is the most widely adopted method, with sprays of fungicides from flowering to pre harvest, and the iprodione products triforine, procymidone, captan, mancozeb are recommended for disease control (May-De Mio et al., 2004). May-De Mio et al. (2011) attributes the preference for the chemical control because of its easiness of use and efficiency to control brown rot, mainly tebuconazole and azoxystrobin. However, the use of the same active ingredient for a few cultivations can promote resistance (Luo & Schnabel, 2008). In spite of all benefits that the fungicide bring to brown rot control, its use is very controversial and it is not allowed during postharvest life because it can harm consumers' health (Adaskaveg & Förster, 2010). During postharvest (storage time), the fungi have an appropriate environment for development and increase brown rot incidence. This fact highlights the need to develop new techniques for brown rot control throughout postharvest.

One technique that requires more studies is the biological control with Trichoderma spp., basically because its use is allowed during the postharvest (storage time). It is an anamorphic fungus found in most types of soil (Harman et al., 2004), the fungus has an antagonistic effect on M. fructicola and can help brown rot control (Hong et al., 1998). Fact that make the use of this fungus successful is its ability to survive within a wide range of temperatures and its broad spectrum action, such as antibiosis, competition and hyperparasitism (Janisiewicz et al., 2000; Howell, 2003; Harman et al., 2004, Brunner et al., 2005). Hong et al., (1998) demonstrated that Trichoderma atroviride and Trichoderma viride have great potential for the control of brown rot in postharvest and reduced between 63-98% incidence in peaches and 67-100% in plums. Nevertheless, the optimal concentration of Trichoderma and its interaction with the fungicides applied before harvest are not clear yet.

Thus, the aim of this research was to evaluate the effect of the application of Trichoderma harzianum Rifai and its association with different fungicides applied during the cultivation (before harvest) of 'Eldorado' peaches on brown rot incidence and other quality parameters during storage.

Material and Methods

This study was conducted in a commercial orchard located in the municipality of Santiago, Rio Grande do Sul, Brazil, and at the Postharvest Research Center of the Federal University of Santa Maria (UFSM). The treatments consisted of five preharvest fungicide applications (control, captan, iprodione, iminoctadine and tebuconazole) associated with postharvest application of T. harzianum after cold storage (with and without application), three evaluation times (zero, two and four days at 20 °C), resulting in a 5x2x3 factorial arrangement (Table 1). The antagonist used was T. harzianum (ETSR 20), obtained from the Phytopathology Laboratory (UFSM). The fungus development was stimulated on rice seeds (Ethur, 2006). To apply the antagonist, a suspension was prepared by washing the rice grain with distillated water. The fungus concentration in this suspension was adjusted to 9 x 10⁷ conidia mL^-1. The peaches treated with T. harzianum were immersed in the antagonist solution for one minute before storage.

Table 1:
Preharvest fungicide application in association with postharvest Trichoderma harzianum application for brown rot incidence control in 'Eldorado', Santa Maria, RS, Brazil

• ^*
  Active ingredient, commercial product, dose and number of applications in preharvest

After storage, the parameters evaluated were: a) occurrence of Monilinia fructicola (Brown rot); b) occurrence of Rhizopus stolonifer (Ehrenb.) Vuill and c) occurrence of Penicillium spp. evaluated by counting the peaches that showed typical fungal lesion and data expressed as percentage of total fruits in the sample; d) Skin browning severity: assessed on a scale of 0 - 3 according to the amount of browning on the surface of the peaches, where 0 = 0% of darkened surface; 1 = > 0% up to 10% of darkened surface; 2 = > 10% up to 30% of darkened surface; 3 = > 30% of darkened surface, according to Brackmann et al. (2009a). The mean was obtained by the total number of peaches multiplied by their respective level of skin browning; this sum was then divided by the total number of peaches in the sample; e) Ethylene production: obtained by gas chromatography. A sample of one kilogram of peaches was hermetically sealed in a 5-L container. After one hour, 1mL of the head space was withdrawn and injected into a chromatograph. Ethylene production was calculated by taking into account ethylene concentration, fruit mass, free room inside the container and time and expressed as µL C₂H₄ kg^-1 h^-1; f) Respiratory rate: obtained by circulating the air of the same container of ethylene production through an electronic gas analyzer: the respiration rate was calculated and expressed as mL CO₂ kg^-1 h^-1.

The experiment was arranged in a 5x2x3 factorial arrangement, with four replications of 25 fruits. Before the analysis of variance (ANOVA), data were submitted to Lilliefors test to check normality. Those data that did not show normal distribution were transformed by the formula ((x + 0,5)/100)^0,5 before the analysis of variance. After this, the means were submitted to Tukey's test with a 5% probability of error.

Results and Discussion

A significant triple interaction between the application of fungicides, T. harzianum and the time evaluation for the parameters incidence of brown rot (Table 2), ethylene production, respiratory rate (Table 4) and skin browning (Table 5), while for the evaluation of the other fungi there was a factorial interaction (Table 3).

Table 2:
Brown rot incidence of 'Eldorado' peaches treated before harvesting with fungicide and stored at -0.5 °C for 40 days and four days at 20 °C in association with Trichoderma harzianum treatment after storage

• ^*
  Means followed by the same small letters in the columns and capital letters in the rows are not significantly different by the Tukey's test at 5% probability.

Table 3:
Incidence of Rhizopus stolonifer and Penicillium spp. in 'Eldorado' peaches treated before harvesting with fungicide and stored at -0.5 °C for 40 days and four days at 20 °C, in association with Trichoderma harzianum treatment after storage

• ^*
  Means followed by the same small letters in the columns and capital letters in the rows are not significantly different by the Tukey's test at 5% probability.

Table 4:
Ethylene production and respiratory rate of 'Eldorado' peaches treated before harvesting with fungicide and stored at -0.5 °C for 40 days and four days at 20 °C, in association with Trichoderma harzianum treatment after storage

• ^*
  Means followed by the same small letters in the columns and capital letters in the rows are not significantly different by the Tukey's test at 5% probability.

Table 5:
Skin browning of 'Eldorado' peaches treated before harvesting with fungicide and stored at -0.5 °C for 40 days and four days at 20 ºC, in association with Trichoderma harzianum treatment after storage

• ^*
  Means followed by the same small letters in the columns and capital letters in the rows are not significantly different by the Tukey's test at 5% probability.

When evaluating the association of fungicide application before harvest with T. harzianumin in the postharvest, the peaches of the control treatment showed higher brown rot incidence, during the whole evaluation, except for the fungicides iprodione and captan, right after the end of storage (zero days at 20°C) (Table 2). No significant difference was observed between the fungicides, up to two days of shelf life, when the peaches received T. harzianum. However, peaches without application of T. harzianum and tebuconazole before harvest had lower brown rot incidence at two days at 20°C. After four days of shelf life at 20°C, the lowest brown rot incidence was verified on peaches with te fungicide iminoctadine associated with the application of T. harzianum in postharvest. This result corroborates the findings of Moreira & May-De Mio (2009), who verified that the application of iminoctadine controlled 96% of brown rot incidence after 6 days of storage at 5 °C followed by 5 days at 20 °C.

Evaluation of the effects of T. harzianum of each treatment showed that its application did not bring benefits for the brown rot control, except for the fungicide captan right after the end of storage (Table 2). However, Hong et al., (1998) obtained 63 - 98% of brown rot control with four isolates of T. harzianum throughout the postharvest. These results differ from those obtained in the present study. This difference can be explained by the concentration of the antagonist used by those authors (10⁸⁾ in comparison with the concentration of 9x10⁷ conidia mL^-1 used in this study.

The application of T. harzianum had no effect on brown rot control; however, a synergic effect with the fungicide application on Rhizopus stolonifer control was observed after four days of shelf life compared with the application of the fungicide alone (Table 3). In fact, T. harzianum showed an effective biological control of the disease during postharvest (Batta, 2007). The same author reported Rhizopus stolonifer control in peaches and strawberries with the application of T. harzianum before incubation at 20 °C. Penicillium spp. did not vary much between treatments; however, it is noteworthy that fruits treated with captan, with or without T. harzianum, had no Penicillium spp. incidence.

Regarding ethylene production, no significant difference was observed between the fungicides, with or without T. harzianum, right after the end of storage (Table 4). After two days of shelf life, higher ethylene production was found in the peaches of the control treatment, probably because of the higher brown rot incidence in the same treatment (Table 2). Another study showed that higher rot incidence culminated in elevated ethylene production in peaches (Brackmann et al., 2009a). After four days, higher ethylene production was observed in the fruit of the control and iminoctadine treatments. Nevertheless, without T. harzianum, the behavior was different and a significant difference was only observed between treatments after four days of shelf life, when the treatment with captan showed higher ethylene production.

Comparing the effect of T. harzianumin in each fungicide treatment, it was found that the antagonist application increased ethylene production significantly in all treatments, after two days of shelf life at 20 °C. This higher ethylene production influenced the fruit negatively, because ethylene triggers a series of events that culminate in fruit ripening. Kiwi fruit infected with Botrytis cinerea showed faster pulp softening as a result of high ethylene production (Brook, 1991). Ethylene is a compound produced by plants and some microorganisms, such as Verticillium spp., Fusarium spp., Colletotrichum spp. and Botrytis cinerea (Tzeng & De Vay, 1984; Kader, 1992; Qadir et al., 1997; Cristescu et al., 2007; Cantu et al., 2009).

Right after the end of storage, the respiration rate was higher in the peaches without fungicide treatment combined with T. harzianum (Table 4). After two and four days of shelf life, the highest respiration rate was verified in the peaches of the control treatment. Similar results were found in the peaches without the application of T. harzianum, where the peaches of the control treatment showed higher respiration rate at chamber opening and four days of shelf life. These results are probably associated with ethylene production of the T. Harzianum fungus, leading to autocatalytic ethylene production, since it is strongly stimulated by exogenous factors, such as fungus infections (Yang & Hoffman, 1984). This process accelerates fruit metabolism and increases respiration and fruit ripening (Chitarra & Chitarra, 2005).

After two days of shelf life, higher skin browning was observed in the peaches treated with T. harzianum as compared to peaches without the fungus treatment, except for peaches with iprodione and T. harzianum (Table 5). Between the fungicides in association with T. harzianum, the fruits of the control treatment showed greater skin browning at chamber opening and differ significantly from the tebuconazole treatment. The evaluation after two days of shelf life revealed greater skin browning of the peaches of the captan treatment, whereas at four days, the highest skin browning level was observed in the peaches of the captan and iminoctadine treatments. This greater skin browning of peaches treated with T. harzianum was probably associated with the growth of the antagonist fungus. Another study suggested that skin browning results from the contact of the skin with water during the application of postharvest fruit treatment (Brackmann et al., 2009b).

Lower incidence of skin browning in fruit treated with iprodione fungicide after two days of evaluation may be due to the high sensitivity of T. harzianum to the active ingredient of the fungicide iprodione. In vitro tests demonstrated the negative effect of iprodione on mycelia development and sporulation of T. harzianum and T. viride (Silva et al., 1999)Another study demonstrated that the same fungicide reduced the development of an antagonist of Trichothecium roseum (Moreira & May De Mio, 2007) by 80%. Among the peaches without T. harzianum treatment, the greatest skin browning was observed in fruits with the captan treatment, after four days of shelf life (Table 5). These results indicated that the T. harzianum and the fungicide captan negatively affected fruit quality with increased skin browning.

Conclusion

The application of T. harzianum during postharvest did not bring benefits to brown rot control, however, the association of T. harzianum and fungicides reduced the incidence of Rhizopus stolonifer during shelf life. The fungicide captan increased skin browning during shelf life, mainly when associated with the application of T. harzianum, but inhibited the incidence of Penicillium spp. The application of T. harzianum in postharvest increased ethylene production in all fungicide treatments of this study.

References

• Adaskaveg JE & Förster H (2010) New developments in postharvest fungicide registrations for edible horticultural crops and use strategies in the United States. In: Prusky D & Gullino ML (Eds.) Postharvest Plant Pathology. Plant Pathology in 21^st century - Contributions to the 9^th International Congress. Dordrecht, Springer Verlag. p.107-117.
• Agrianual (2011) Anuário da Agricultura Brasileira. São Paulo, Editora FNP. 520p.
• Batta YA (2007) Control of postharvest diseases of fruit with an invert emulsion formulation of Trichoderma harzianum Rifai. Postharvest Biology and Technology, 43:143-150.
• Brackmann A, Pavanello EP, Both V, Weber A & Pinto JAV (2009a) Atmosfera refrigerada e controlada para pêssegos 'Eragil'. Ciência Rural, 39:2010-2015.
• Brackmann A, Weber A, Giehl RFH & Eisermann AC (2009b) Pré-resfriamento sobre a qualidade de pêssegos 'Chiripá'. Ciência Rural, 39:2354-2360.
• Brook PJ (1991) Botrytis stemend rot and other storage diseases of kiwifruit a review. Acta Horticulture, 297:545-550.
• Brunner K, Zeilinger S, Ciliento R, Woo SL, Lorito M, Kubicek CP & Mach RL (2005) Genetic improvement of a fungal biocontrol agent to enhance both antagonism and induction of plant systemic disease resistance. Applied and Environmental Microbiology, 71:3959-3965.
• Cantu D, Blanco-Ulate B, Yang L, Labavitch JM, Bennett AB & Powell ALT (2009) Ripening-Regulated Susceptibility of Tomato Fruit to Botrytis cinerea Requires NOR But Not RIN or Ethylene. Plant Physiology, 150:1434-1449.
• Chitarra MIF & Chitarra AB (2005) Pós-colheita de frutos e hortaliças: fisiologia e manuseio. 2ª ed. Lavras, UFLA. 785p.
• Cristescu SM, Harrem F & Woltering E (2007) Ethylene production of Botrytis cinerea in vitro and during in plant infection of tomato fruits. Advances in Plant Ethylene Research, 6:395-397.
• Ethur LZ (2006) Dinâmica populacional e ação de Trichoderma no controle de fusariose em mudas de tomateiro e pepineiro. Tese de Doutorado. Universidade Federal de Santa Maria, Santa Maria. 154p.
• FAO - Food and Agriculture Organization of the United Nations (2012). Faostat. Disponível em: Disponível em: http://faostat.fao.org/site Acessado em: 01 de maio de 2012.
  » http://faostat.fao.org/site
• Harman GE, Howell CR, Viterbo A, Chet I & Lorito M (2004) Trichoderma species - opportunistic, avirulent plant symbionts. Nature, 2:43-56.
• Howell CR (2003) Mechanisms employed by Trichoderma species in the biological control of plant disease: the history and evolution of current concepts. Plant Disease, 87:04-10.
• Hong CX, Michailides TJ & Holtz BA (1998) Effects of wounding, inoculum density, and biological control agents on postharvest Brown rot of stone fruit. Plant Disease, 82:1210-1216.
• Janisiewicz WJ, Tworkoski TJ & Sharer C (2000) Characterizing the mechanism of biological control of postharvest diseases on fruits with a simple method to study competition for nutrients. Phytopathology, 90:1196-1200.
• Kader AA (1992): Postharvest Biology and Technology an overview. In: Kader AA (Ed.) Postharvest Technology of Horticultural Crops. Davis, University of California. 296 p. (Publication, 3311).
• Luo CX & Schnabel G (2008) Adaptation to fungicides in Monilinia fructicola isolates with different fungicide resistance phenotypes. Phytopathology, 98:230-238.
• May-DeMio LL, Garrido L & Ueno B (2004) Doenças de fruteiras de caroço. In: Monteiro LB, May-De Mio LL, Serrat BM, Motta AC & Cuquel FL (2004) Fruteiras de caroço: uma visão ecológica. Curitiba, UFPR. p.169-221.
• May-De Mio LL, Luo Y & Michailides TJ (2011) Sensitivity of Monilinia fructicola from Brazil to tebuconazole, azoxystrobin, thiophanate-methyl and implications for disease management. Plant Disease, 95:821-827.
• Moreira LM & May-De Mio LM (2007) Crescimento micelial de Monilinia fructicola e Trichothecium roseum em diferentes temperaturas e sensibilidade do antagonista a fungicidas e fosfitos. Scientia Agrária, 8:337-341.
• Moreira LM & May-De Mio LM (2009) Controle da podridão parda do pessegueiro com fungicidas e fosfitos avaliados em pré e pós-colheita. Ciência e Agrotecnologia, 33:405-411.
• Qadir A, Hewett EW & Long PG (1997) Ethylene production by Botrytis cinerea Postharvest Biology and Technology, 11:85-91.
• Silva ACF, Rosa CRA & Melo IS (1999) Sensibilidade de isolados de Trichoderma spp. a benomil e iprodiona. Revista Científica do Centro de Ciências Rurais, 3:395-399.
• Tzeng DD & De Vay JE (1984) Ethylene production and toxicity of methionine and its derivatives with riboflavin in cultures of Verticillium, Fusarium and Colletotrichum species to light. Physiologia Plantarum, 62:545-552.
• Yang SF & Hoffman NE (1984) Ethylene biosynthesis and its regulation in higher plants. Annual Review of Plant Physiology, 35:155-189.

Publication Dates

• Publication in this collection
  Dec 2015

History

• Received
  04 Aug 2014
• Accepted
  28 Sept 2015