The efficacy of selected biological control agents against citrus black spot ( CBS ) pathogen Phyllosticta citricarpa

Citrus black spot (CBS) caused by Phyllosticta citricarpa (McAlpine) Van der Aa (asexual state) synonym Guignardia citricarpa Kiely (sexual state) is one of the most devastating diseases of citrus which occurs in various citrus producing areas around the globe. Management is mainly based on monthly applications of copper fungicides and strobilurins under field conditions. In this study, biocontrol agents were evaluated as alternative post-harvest treatments against citrus black spot disease. Two bio-control agents namely, Bacillus subtilis Cohn and Trichoderma harzianum Rifai were evaluated for their efficacy against P. citricarpa. Their efficacy was further compared with commercial fungicides Dithane 750 (Mancozeb, 750 g/kg). Results obtained showed that T. harzianum treatments were highly suppressive towards pathogen growth in vitro (85%) as compared to B. subtillis (4%) and Dithane 750 (12%). Treatment of artificially inoculated fruits with combined formulations of both biocontrol agents resulted in reduced CBS severity as compared to their single applications. These findings suggest that T. harzianum had highest suppression of pathogen growth as compared to B. subtilis. The results also suggest that their combined application can provide an effective disease management when compared to sole application of Dithane 750. Further studies are however, needed to determine their effectiveness under field conditions and also their efficacy can be sustained when applied with commercial fungicides.


INTRODUCTION
Citrus black spot caused by Phyllosticta citricarpa (McAlpine) van der Aa is a major disease affecting citrus production worldwide, where weather conditions are favourable (Paul et al., 2005).The disease affects all citrus species of economic importance however the disease causes more damage to sweet oranges and *Corresponding author.E-mail: Mapotso.kena@ul.ac.za.Tel: +27 15 2682927.Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License lemons due to their high susceptibility (Kotźe, 1981).The disease is of high economic importance in citrus growing countries including South Africa (Smith et al., 1997).The causal pathogen causes external blemishes on the fruit rind, making the fruit unsuitable for fresh market which in some cases, result in severe fruit drop and crop losses (Araújo et al., 2013).Worldwide, the potential economic loss is mainly due to the rejection from fresh market as a result of unsightly lesions (Paul et al., 2005;Everett and Rees-George, 2006).Potential quarantine of fruit sales to other citrus production regions can also result in economic losses of millions of dollars (Meyer et al., 2012).
Citrus black spot management relies mainly on the application of preventative fungicides during fruit susceptibility (Schutte et al., 1997).Various groups of fungicides have been used against citrus black spot with varying degree of efficacy (Rodrigues et al., 2007).However, loss of sensitivity and pathogen resistance is a common factor resulting in poor disease control by most fungicides groups (Akinnifesi et al., 2006;Rodrigues et al., 2007).Growing international concern on chemical residues on treated fruits has also resulted in need to identify and develop alternative safe measures.Biological control agents such as Trichoderma spp.and Bacillus spp.have been shown to be effective against a number of plant pathogens (Kupper et al., 2011;Abdalla et al., 2014).
Also, combined application of biological control agents has been shown to be effective against some plant diseases (Begum et al., 2010).There is limited information regarding the use of both Trichoderma.harzianum strain and Bacillus subtilis in the suppression of pathogen growth in vitro and disease severity in vivo.
The main aim of this study was to evaluate the combined efficacy of biological control agents (T.harzianum and B. subtilis) in comparison with commercial fungicides (Dithane 750) against CBS pathogen P. citricarpa.
The specific objectives were, to determine the effect of B. subtilis and T. harzianum, as compared to Dithane 750 on P. citricarpa growth in vitro and to investigate the effectiveness of both biological control agents on citrus black spot development, when applied separately or in combination.

Microbial culture preparations and maintenance
A pure culture of P. citricarpa isolate (PPRI 8774) was obtained from the National Fungal Collection, Biosystematics Division, Agricultural Research Council-Plant Protection Research Institute (ARC-PPRI), Pretoria, South Africa.The pathogen was originally isolated from an infected 'Valencia' citrus fruits from Letsetele farm in Limpopo, South Africa.
The isolate was preserved on 2% Potato dextrose agar (PDA) and stored at 4°C until further use.The pathogen was identified as P. citricarpa using both morphological and molecular characterization.Molecular characterization was based on Polymerase Chain Reaction (PCR) technique described by Perez et al. (2007).Two biological agents namely T. harzianum (PPRI 8230) and B. subtilis (B246) were obtained from ARC-Plant Protection Research Institute and Quality Management Services (QMS) Company at Tzaneen area in Limpopo Province, South Africa, respectively.Both B. subtilis and T. harzianum were maintained in Nutrient Agar and 2% PDA, respectively and kept at 4°C until further use.

Effect of biocontrol agents on P. citricarpa growth in vitro
The growth inhibition of P. citricarpa by T. harzianum and B. subtilis was carried out on Potato Dextrose Agar (PDA) and Nutrient Agar (NA), respectively, using the dual culture technique.This was determined by placing 5 mm mycelial plug obtained from a 7 days old P. citricarpa culture, at 1 cm from the periphery of 90 mm Petri plates with PDA and incubated for 3 days at ± 28°C (Evans et al., 2003).After 3 days, a 5 mm mycelial plug obtained from 7 days old T. harzianum culture was placed 1 cm away from the edge, of the same Petri dish on the side of P. citricarpa.Control treatments were not inoculated with a biological control agent.
All plates were incubated for 7 days at ± 26°C.In B. subtilis treatments, dual culture method described by Etebarian et al. (2005) was followed.Half of the petri plates containing nutrient agar were streaked with 100 ml suspension of B. subtilis and incubated for 48 h.Thereafter, 5 mm mycelial plug of 7 days old P. citricarpa culture was inoculated as described above.
Fungicide solution was prepared by mixing 7.5 g of Dithane 750 with 100 ml sterile distilled water and kept at 4°C until further use (concentration).Used fungicide dilutions were according to manufacturer's instructions.For pathogen suppression, 10 ml of prepared suspension of Dithane 750 was added to 1 litre PDA after autoclaving at 40°C and dispensed into 90 mm petri plates.Each treatment was replicated four times and experiment was repeated twice.
For in vivo evaluation, a total of 200 healthy Valencia fruits were harvested from Letsetele citrus farm, surface sterilised with 0.1% Sodium hypochloride for 3 min, rinsed with sterile distilled water twice and air dried at room temperature for 24 h.Inoculum preparation and fruit inoculation was done according to the method described by Baldassari et al. (2009).Mature Valecia orange leaves were used for pathogen inoculum production.A 10 mm leave disk was cut with cork borer and placed on ripe orange fruit.Inoculated fruits were incubated at ± 25°C for 21 days after which lesion size was measured using a Venier caliper.
Bio-control treatments were prepared by mixing 50:50 suspensions of B. subtilis (1 × 10 7 cells/mL) and T. harzianum culture filtrate (5 × 10 5 spores/Ml) and applied separately or in combination 24 h after fruits were inoculated with pathogen.For control, inoculated fruits were sprayed with sterile distilled water.All treatments were laid out in a completely randomized design (CRD) with five replicates.Disease severity was evaluated on a scale of 0 to 4 where 0= no lesion, 1 = 10% fruit area affected, 2 = 10 to 25% fruit area affected, 3 = 25 to 30% fruit area affected, 4 ≥ 50% area affected.Collected data was analysed using Statistic 10.0 software.Duncan's Multiple Range test was used to compare treatment means at P≤ 0.05 probability level.

RESULTS AND DISCUSSION
Results for in vitro experiment are presented in Figure 1.The table is not correct.The treatments comparison is wrong because there are a lot of variations (culture medium, organisms, fungicide, seeding of the organism, etc.).The treatments can be compared with its control, but not with each other Dual culture test results which showed highest pathogen growth inhibition occurring in T. harzianum treatments at 85%.P. citricarpa growth inhibition was lowest in B. subtilis treatment at 4%.The same results regarding the suppressive effect of B. subtilis was also observed in inoculated fruits (Table 1), where lesion size was significantly higher than T. harzianum treatments at 1.3 and 0.2 mm, respectively.However, the level of inhibition was not significantly different in comparison with Dithane 750.Dithane 750 is currently one of the fungicides used in the management of Citrus black spot (CBS), however loss of pathogen sensitivity is a concern (Kupper et al., 2011).
Various reports have shown positive efficiency of T. harzianum in the management of other plant diseases (Thilagavathi et al., 2007;Begum et al., 2010).The same results was also observed in our study where T. harzianum was able to significantly suppress the growth P. citricarpa in vitro and also reduce disease severity in artificially infected citrus fruits (Table 2).Treatment of infected fruits with B. subtilis also resulted in a significantly high number of fruits, showing disease symptoms and severity (1.8).
However, when B. subtilis was applied in combination with T. harzianum, a significant reduction in disease severity was observed.In previous reports, application of strain mixture of B. subtilis, Pseudomonas and Trichoderma spp.resulted in an increased suppression of B. cinerea than when each biocontrol agent was applied alone (Thilagavathi et al., 2007).It has been suggested by various authors that when applying biocontrol agents as combination in disease management, they should be compatible to achieve consistent control (Raaijmakers et al., 1996).
In our study, results show that treatment of CBS infected fruits with a combination of both T. harzianum and B. subtilis formulations significantly reduced number of citrus, showing CBS symptoms which also reduced disease severity (Table 2).Citrus fruit treatment with both T. harzianum and B. subtilis significantly reduced the number of diseased plants and suppressed disease symptom appearance.This combined effect corresponds with previous findings where combined application of B. subtilis and yeast isolates reduced gray mould in treated apple fruits (Zangoei et al., 2014).Considering the good performance of both biocontrol when used in combination, it is anticipated that their use against citrus black spot could be beneficial when applied as a postharvest treatment.

Figure 1 .
Figure 1.Inhibitory effect of bio-control agents and fungicide on P. citricarpa after 30 days of inoculation.

Table 2 .
Effects of T. harzianum and B. subtilis applied separately or in combination on CBS occurrence and severity.
a Disease severity was rated on a scale of 0 to 4. b Numbers within a same column followed by the same letter are not significantly different according duncan's multiple range tests.