Investigating the role of mycorrhizal fungi and associated bacteria in promoting growth of citrus seedlings
- Authors: Sitole, Phumeza
- Date: 2014
- Subjects: Mycorrhizal fungi , Citrus -- South Africa , Citrus -- Diseases and pests -- Biological control -- South Africa , Fungi as biological pest control agents , Bacteria , Phytophthora , Pythium , Indoleacetic acid
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4111 , http://hdl.handle.net/10962/d1013033
- Description: South Africa is the world's second largest exporter of fresh citrus and is ranked 14th in citrus production. Fungal pathogens such as Phytophthora and Pythium cause economic losses as a result of root rot and brown rot. Mycorrhizal fungi are specialized members of the fungal community forming a mutualistic relationship with plant roots. Mycorrhizal fungal structures are known to associate with other soil microorganisms and these may contribute to improved plant growth. A diverse group of bacteria that interact with the mycorrhizal fungi are known as Mycorrhizal Helper Bacteria (MHB). The aim of this study was to investigate the role of arbuscular mycorrhiza and associated bacteria isolated from spores and determine whether they had any plant growth promoting potential. A total of 19 bacteria were isolated from arbuscular mycorrhizal spores and were molecularly identified as belonging to several Bacillus, Micrococcus, Onchrobactrum and Staphylococcus sp. All bacterial isolates were tested for plant growth promotion abilities. One Bacillus isolate was able to solubilise phosphate. Four isolates Micrococcus sp, Micrococcus leteus, Ochrobacterum sp and Ochrobacterum antropi were able to produce Indole Acetic Acid and three isolates showed potential to reduce growth of Phytophthora nicotianae, P. citrocola and P. citrophthora in in vitro plate cultures. Further tests using culture supernatants of the Bacillus sp, Micrococcus sp and Bacillus cereus confirmed their ability to inhibit or reduce growth of the three Phytophthora species in a 96 well bioassay. Bacillus sp and Bacillus cereus were able to inhibit Phytophthora spp by 95 to 100 % and Micrococcus spp was able to decrease pathogen growth by 60 to 94 %. These bacterial isolates were further evaluated for plant growth promoting abilities on citrus rough lemon seedlings alone or in combination with arbuscular mycorrhizal inoculum. Bacterial and mycorrhizal inoculants influence the increase in shoot and root biomass. Bacillus cereus in combination with mycorrhizal inoculum significantly increased seedling shoot to root ratio while root biomass was significantly increased with mycorrhizal inoculation. Due to the short duration of the trial mycorrhizal colonisation could not be assessed. It is evident that selected combinations of bacteria and mycorrhizal fungi could promote citrus seedling growth and potentially improve seedling health. Further studies under nursery conditions are recommended.
- Full Text:
- Date Issued: 2014
- Authors: Sitole, Phumeza
- Date: 2014
- Subjects: Mycorrhizal fungi , Citrus -- South Africa , Citrus -- Diseases and pests -- Biological control -- South Africa , Fungi as biological pest control agents , Bacteria , Phytophthora , Pythium , Indoleacetic acid
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4111 , http://hdl.handle.net/10962/d1013033
- Description: South Africa is the world's second largest exporter of fresh citrus and is ranked 14th in citrus production. Fungal pathogens such as Phytophthora and Pythium cause economic losses as a result of root rot and brown rot. Mycorrhizal fungi are specialized members of the fungal community forming a mutualistic relationship with plant roots. Mycorrhizal fungal structures are known to associate with other soil microorganisms and these may contribute to improved plant growth. A diverse group of bacteria that interact with the mycorrhizal fungi are known as Mycorrhizal Helper Bacteria (MHB). The aim of this study was to investigate the role of arbuscular mycorrhiza and associated bacteria isolated from spores and determine whether they had any plant growth promoting potential. A total of 19 bacteria were isolated from arbuscular mycorrhizal spores and were molecularly identified as belonging to several Bacillus, Micrococcus, Onchrobactrum and Staphylococcus sp. All bacterial isolates were tested for plant growth promotion abilities. One Bacillus isolate was able to solubilise phosphate. Four isolates Micrococcus sp, Micrococcus leteus, Ochrobacterum sp and Ochrobacterum antropi were able to produce Indole Acetic Acid and three isolates showed potential to reduce growth of Phytophthora nicotianae, P. citrocola and P. citrophthora in in vitro plate cultures. Further tests using culture supernatants of the Bacillus sp, Micrococcus sp and Bacillus cereus confirmed their ability to inhibit or reduce growth of the three Phytophthora species in a 96 well bioassay. Bacillus sp and Bacillus cereus were able to inhibit Phytophthora spp by 95 to 100 % and Micrococcus spp was able to decrease pathogen growth by 60 to 94 %. These bacterial isolates were further evaluated for plant growth promoting abilities on citrus rough lemon seedlings alone or in combination with arbuscular mycorrhizal inoculum. Bacterial and mycorrhizal inoculants influence the increase in shoot and root biomass. Bacillus cereus in combination with mycorrhizal inoculum significantly increased seedling shoot to root ratio while root biomass was significantly increased with mycorrhizal inoculation. Due to the short duration of the trial mycorrhizal colonisation could not be assessed. It is evident that selected combinations of bacteria and mycorrhizal fungi could promote citrus seedling growth and potentially improve seedling health. Further studies under nursery conditions are recommended.
- Full Text:
- Date Issued: 2014
Entomopathogenic fungi for control of soil-borne life stages of false codling moth, Thaumatotibia leucotreta (Meyrick) (1912) (Lepidoptera: Tortricidae)
- Authors: Coombes, Candice Anne
- Date: 2013
- Subjects: Tortricidae , Lepidoptera , Cryptophlebia leucotreta , Insect pests -- Biological control -- South Africa -- Eastern Cape , Tortricidae -- Biological control -- South Africa -- Eastern Cape , Citrus -- Diseases and pests -- Biological control -- South Africa -- Eastern Cape , Entomopathogenic fungi , Fungi as biological pest control agents , Biological pest control agents
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5607 , http://hdl.handle.net/10962/d1002057 , Tortricidae , Lepidoptera , Cryptophlebia leucotreta , Insect pests -- Biological control -- South Africa -- Eastern Cape , Tortricidae -- Biological control -- South Africa -- Eastern Cape , Citrus -- Diseases and pests -- Biological control -- South Africa -- Eastern Cape , Entomopathogenic fungi , Fungi as biological pest control agents , Biological pest control agents
- Description: False codling moth (FCM), Thaumatotibia leucotreta is an extremely important pest of citrus in South Africa and with the shift away from the use of chemicals, alternate control options are needed. One avenue of control which has only recently been investigated against the soil-borne life stages of FCM is the use of entomopathogenic fungi (EPF). In 2009, 12 entomopathogenic fungal isolates collected from South African citrus orchards showed good control potential during laboratory conducted bioassays. The aim of this study was to further analyse the potential of these isolates through concentration-dose and exposure-time response bioassays. After initial re-screening, concentration-dose response and exposure-time response sandconidial bioassays, three isolates were identified as exhibiting the greatest control potential against FCM in soil, Metarhizium anisopliae var. anisopliae (G 11 3 L6 and FCM Ar 23 B3) and Beauveria bassiana (G Ar 17 B3). Percentage mycosis was found to be directly related to fungal concentration as well as the amount of time FCM 5th instar larvae were exposed to the fungal conidia. LC50 values for the three isolates were not greater than 1.92 x 10⁶ conidia.ml⁻ₑ and at the LC₅₀, FCM 5th instar larvae would need to be exposed to the fungus for a maximum of 13 days to ensure a high mortality level. These isolates along with two commercially available EPF products were subjected to field persistence trials whereby net bags filled with a mixture of autoclaved sand and formulated fungal product were buried in an Eastern Cape citrus orchard. The viability of each isolate was measured on a monthly basis for a period of six months. All isolates were capable of persisting in the soil for six months with the collected isolates persisting far better than the commercially used isolates. Two of the isolates, G 11 3 L6 and G Ar 17 B3, were subjected to small scale laboratory application trials. Two formulations were investigated at two concentrations. For each isolate, each formulation and each concentration, FCM 5th instar larvae were applied and allowed to burrow into the soil to pupate before fungal application or after fungal application. Contact between fungi and FCM host is essential as, in contrast to pre-larval treatments, percentage mortality in post-larval treatments was low for both formulations and both isolates. For isolate G Ar 17 B3, a conidial suspension applied as a spray at a concentration of 1 x 10⁷ conidia.ml⁻ₑ obtained the highest percentage mortality (80 %). For isolate G 11 3 L6 however, both formulations performed equally well at a high, 1 x10⁷ conidia.ml⁻ₑ concentration (conidial suspension: 60 %; granular: 65 %) The results obtained thus far are promising for the control of FCM in citrus, but if these EPFs are to successfully integrate into current FCM control practices more research, some of which is discussed, is essential
- Full Text:
- Date Issued: 2013
- Authors: Coombes, Candice Anne
- Date: 2013
- Subjects: Tortricidae , Lepidoptera , Cryptophlebia leucotreta , Insect pests -- Biological control -- South Africa -- Eastern Cape , Tortricidae -- Biological control -- South Africa -- Eastern Cape , Citrus -- Diseases and pests -- Biological control -- South Africa -- Eastern Cape , Entomopathogenic fungi , Fungi as biological pest control agents , Biological pest control agents
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5607 , http://hdl.handle.net/10962/d1002057 , Tortricidae , Lepidoptera , Cryptophlebia leucotreta , Insect pests -- Biological control -- South Africa -- Eastern Cape , Tortricidae -- Biological control -- South Africa -- Eastern Cape , Citrus -- Diseases and pests -- Biological control -- South Africa -- Eastern Cape , Entomopathogenic fungi , Fungi as biological pest control agents , Biological pest control agents
- Description: False codling moth (FCM), Thaumatotibia leucotreta is an extremely important pest of citrus in South Africa and with the shift away from the use of chemicals, alternate control options are needed. One avenue of control which has only recently been investigated against the soil-borne life stages of FCM is the use of entomopathogenic fungi (EPF). In 2009, 12 entomopathogenic fungal isolates collected from South African citrus orchards showed good control potential during laboratory conducted bioassays. The aim of this study was to further analyse the potential of these isolates through concentration-dose and exposure-time response bioassays. After initial re-screening, concentration-dose response and exposure-time response sandconidial bioassays, three isolates were identified as exhibiting the greatest control potential against FCM in soil, Metarhizium anisopliae var. anisopliae (G 11 3 L6 and FCM Ar 23 B3) and Beauveria bassiana (G Ar 17 B3). Percentage mycosis was found to be directly related to fungal concentration as well as the amount of time FCM 5th instar larvae were exposed to the fungal conidia. LC50 values for the three isolates were not greater than 1.92 x 10⁶ conidia.ml⁻ₑ and at the LC₅₀, FCM 5th instar larvae would need to be exposed to the fungus for a maximum of 13 days to ensure a high mortality level. These isolates along with two commercially available EPF products were subjected to field persistence trials whereby net bags filled with a mixture of autoclaved sand and formulated fungal product were buried in an Eastern Cape citrus orchard. The viability of each isolate was measured on a monthly basis for a period of six months. All isolates were capable of persisting in the soil for six months with the collected isolates persisting far better than the commercially used isolates. Two of the isolates, G 11 3 L6 and G Ar 17 B3, were subjected to small scale laboratory application trials. Two formulations were investigated at two concentrations. For each isolate, each formulation and each concentration, FCM 5th instar larvae were applied and allowed to burrow into the soil to pupate before fungal application or after fungal application. Contact between fungi and FCM host is essential as, in contrast to pre-larval treatments, percentage mortality in post-larval treatments was low for both formulations and both isolates. For isolate G Ar 17 B3, a conidial suspension applied as a spray at a concentration of 1 x 10⁷ conidia.ml⁻ₑ obtained the highest percentage mortality (80 %). For isolate G 11 3 L6 however, both formulations performed equally well at a high, 1 x10⁷ conidia.ml⁻ₑ concentration (conidial suspension: 60 %; granular: 65 %) The results obtained thus far are promising for the control of FCM in citrus, but if these EPFs are to successfully integrate into current FCM control practices more research, some of which is discussed, is essential
- Full Text:
- Date Issued: 2013
Investigation of entomopathogenic fungi for control of false codling moth, Thaumatotibia leucotrata, Mediterranean fruit fly, Ceratitis capitata and Natal fruit fly, C. rosa in South African citrus
- Authors: Goble, Tarryn Anne
- Date: 2010
- Subjects: Insect pests -- Biological control , Tortricidae -- Biological control -- South Africa , Tephritidae -- Biological control -- South Africa , Citrus -- Diseases and pests -- Biological control -- South Africa , Entomopathogenic fungi , Fungi as biological pest control agents , Biological pest control agents
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5723 , http://hdl.handle.net/10962/d1005409 , Insect pests -- Biological control , Tortricidae -- Biological control -- South Africa , Tephritidae -- Biological control -- South Africa , Citrus -- Diseases and pests -- Biological control -- South Africa , Entomopathogenic fungi , Fungi as biological pest control agents , Biological pest control agents
- Description: The biology of key citrus pests Thaumatotibia leucotreta Meyrick (Lepidoptera: Tortricidae), Ceratitis capitata Wiedemann (Diptera: Tephritidae) and Ceratitis rosa Karsch (Diptera: Tephritidae) includes their dropping from host plants to pupate in the soil below citrus trees. Since most EP fungi are soil-borne microorganisms, the development and formulation of alternative control strategies using these fungi as subterranean control agents, targeted at larvae and pupae in the soil, can potentially benefit existing IPM management of citrus in South Africa. Thus, a survey of occurrence of entomopathogenic fungi was undertaken on soils from citrus orchards and natural vegetation (refugia) on conventionally and organically managed farms in the Eastern Cape Province in South Africa. A method for baiting soil samples with citrus pest T. leucotreta and C. capitata larvae, as well as with the standard bait insect, Galleria mellonella Linnaeus (Lepidoptera: Pyralidae), was implemented. Sixty-two potentially useful entomopathogenic fungal isolates belonging to four genera were collected from 288 soil samples, an occurrence frequency of 21.53%. The most frequently isolated entomopathogenic fungal species was Beauveria bassiana (Balsamo) Vuillemin (15.63%), followed by Metarhizium anisopliae var. anisopliae (Metschnikoff) Sorokin (3.82%). Galleria mellonella was the most effective insect used to isolate fungal species (χ2=40.13, df=2, P≤ 0.005), with a total of 45 isolates obtained, followed by C. capitata with 11 isolates, and T. leucotreta with six isolates recovered. There was a significantly (χ2=11.65, df=1, P≤ 0.005) higher occurrence of entomopathogenic fungi in soil samples taken from refugia compared to cultivated orchards of both organically and conventionally managed farms. No significant differences were observed in the recovery of fungal isolates when soil samples from both farming systems were compared. The physiological effects and host range of 21 indigenous fungal isolates obtained in the Eastern Cape were investigated in the laboratory to establish whether these isolates could be effectively used as biological control agents against the subterranean life stages of C. rosa, C. capitata and T. leucotreta. When these pests were treated with a fungal concentration of 1 x 10⁷ conidia ml⁻¹, the percentage of T. leucotreta adults which emerged in fungal treated sand ranged from 5 to 60% (F=33.295; df=21; P=0.0001) depending on fungal isolate and the percentage of pupae with visible signs of mycosis ranged from 21 to 93% (F= 96.436; df=21; P=0.0001). Based on fungal isolates, the percentage adult survival in C. rosa and C. capitata ranged from 30 to 90% and 55 to 86% respectively. The percentage of C. rosa and C. capitata puparia with visible signs of mycosis ranged from 1 to 14% and 1 to 11% respectively. Deferred mortality due to mycosis in C. rosa and C. capitata adult flies ranged from 1 to 58% and 1 to 33% respectively, depending on fungal isolate. Entomopathogenic fungal isolates had a significantly greater effect on the adults of C. rosa and C. capitata than they did on the puparia of these two fruit fly species. Further, C. rosa and C. capitata did not differ significantly in their response to entomopathogenic fungi when adult survival or adult and pupal mycosis were considered. The relative potency of the four most virulent Beauveria isolates as well as the commercially available Beauveria bassiana product, Bb Plus® (Biological Control Products, South Africa), were compared against one another as log-probit regressions of mortality against C. rosa, C. capitata and T. leucotreta which all exhibited a dose-dependent response. Against fruit flies the estimated LC50 values of all five Beauveria isolates ranged from 5.5 x 10¹¹ to 2.8 x 10¹² conidia/ml⁻¹. There were no significant differences between the relative potencies of these five fungal isolates. When T. leucotreta was considered, isolates: G Moss R10 and G 14 2 B5 and Bb Plus® were significantly more pathogenic than G B Ar 23 B3 and FCM 10 13 L1. The estimated LC₅₀ values of the three most pathogenic isolates ranged from 6.8 x 10⁵ to 2.1 x 10⁶ conidia/ml⁻¹, while those of the least pathogenic ranged from 1.6 x 10⁷ to 3.7 x 10⁷ conidia/ml⁻¹. Thaumatotibia leucotreta final instar larvae were exposed to two conidial concentrations, at four different exposure times (12, 48, 72 and 96 hrs) and showed an exposure time-dependant relationship (F=5.43; df=3; P=0.001). At 1 x 10⁷conidia/ml⁻¹ two Beauveria isolates: G Moss R10 and G 14 2 B5 were able to elicit a response in 50% of test insects at 72 hrs (3 days) exposure. Although a limited amount of mycosis was observed in the puparia of both fruit fly species, deferred adult mortality due to mycosis was high. The increased incidence of adult mortality suggests that post emergence mycosis in adult fruit flies may play a more significant role in field suppression than the control of fruit flies at the pupal stage. The increased incidence of pupal mortality, as well as the relatively low concentrations of conidia required to elicit meaningful responses in T. leucotreta pupae may suggest that pre-emergent control of false codling moth will play a more significant role in field suppression than the control of adult life stages using indigenous isolates of entomopathogenic fungi. Various entomopathogenic fungal application techniques targeted at key insect pests within integrated pest management (IPM) systems of citrus are discussed.
- Full Text:
- Date Issued: 2010
- Authors: Goble, Tarryn Anne
- Date: 2010
- Subjects: Insect pests -- Biological control , Tortricidae -- Biological control -- South Africa , Tephritidae -- Biological control -- South Africa , Citrus -- Diseases and pests -- Biological control -- South Africa , Entomopathogenic fungi , Fungi as biological pest control agents , Biological pest control agents
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5723 , http://hdl.handle.net/10962/d1005409 , Insect pests -- Biological control , Tortricidae -- Biological control -- South Africa , Tephritidae -- Biological control -- South Africa , Citrus -- Diseases and pests -- Biological control -- South Africa , Entomopathogenic fungi , Fungi as biological pest control agents , Biological pest control agents
- Description: The biology of key citrus pests Thaumatotibia leucotreta Meyrick (Lepidoptera: Tortricidae), Ceratitis capitata Wiedemann (Diptera: Tephritidae) and Ceratitis rosa Karsch (Diptera: Tephritidae) includes their dropping from host plants to pupate in the soil below citrus trees. Since most EP fungi are soil-borne microorganisms, the development and formulation of alternative control strategies using these fungi as subterranean control agents, targeted at larvae and pupae in the soil, can potentially benefit existing IPM management of citrus in South Africa. Thus, a survey of occurrence of entomopathogenic fungi was undertaken on soils from citrus orchards and natural vegetation (refugia) on conventionally and organically managed farms in the Eastern Cape Province in South Africa. A method for baiting soil samples with citrus pest T. leucotreta and C. capitata larvae, as well as with the standard bait insect, Galleria mellonella Linnaeus (Lepidoptera: Pyralidae), was implemented. Sixty-two potentially useful entomopathogenic fungal isolates belonging to four genera were collected from 288 soil samples, an occurrence frequency of 21.53%. The most frequently isolated entomopathogenic fungal species was Beauveria bassiana (Balsamo) Vuillemin (15.63%), followed by Metarhizium anisopliae var. anisopliae (Metschnikoff) Sorokin (3.82%). Galleria mellonella was the most effective insect used to isolate fungal species (χ2=40.13, df=2, P≤ 0.005), with a total of 45 isolates obtained, followed by C. capitata with 11 isolates, and T. leucotreta with six isolates recovered. There was a significantly (χ2=11.65, df=1, P≤ 0.005) higher occurrence of entomopathogenic fungi in soil samples taken from refugia compared to cultivated orchards of both organically and conventionally managed farms. No significant differences were observed in the recovery of fungal isolates when soil samples from both farming systems were compared. The physiological effects and host range of 21 indigenous fungal isolates obtained in the Eastern Cape were investigated in the laboratory to establish whether these isolates could be effectively used as biological control agents against the subterranean life stages of C. rosa, C. capitata and T. leucotreta. When these pests were treated with a fungal concentration of 1 x 10⁷ conidia ml⁻¹, the percentage of T. leucotreta adults which emerged in fungal treated sand ranged from 5 to 60% (F=33.295; df=21; P=0.0001) depending on fungal isolate and the percentage of pupae with visible signs of mycosis ranged from 21 to 93% (F= 96.436; df=21; P=0.0001). Based on fungal isolates, the percentage adult survival in C. rosa and C. capitata ranged from 30 to 90% and 55 to 86% respectively. The percentage of C. rosa and C. capitata puparia with visible signs of mycosis ranged from 1 to 14% and 1 to 11% respectively. Deferred mortality due to mycosis in C. rosa and C. capitata adult flies ranged from 1 to 58% and 1 to 33% respectively, depending on fungal isolate. Entomopathogenic fungal isolates had a significantly greater effect on the adults of C. rosa and C. capitata than they did on the puparia of these two fruit fly species. Further, C. rosa and C. capitata did not differ significantly in their response to entomopathogenic fungi when adult survival or adult and pupal mycosis were considered. The relative potency of the four most virulent Beauveria isolates as well as the commercially available Beauveria bassiana product, Bb Plus® (Biological Control Products, South Africa), were compared against one another as log-probit regressions of mortality against C. rosa, C. capitata and T. leucotreta which all exhibited a dose-dependent response. Against fruit flies the estimated LC50 values of all five Beauveria isolates ranged from 5.5 x 10¹¹ to 2.8 x 10¹² conidia/ml⁻¹. There were no significant differences between the relative potencies of these five fungal isolates. When T. leucotreta was considered, isolates: G Moss R10 and G 14 2 B5 and Bb Plus® were significantly more pathogenic than G B Ar 23 B3 and FCM 10 13 L1. The estimated LC₅₀ values of the three most pathogenic isolates ranged from 6.8 x 10⁵ to 2.1 x 10⁶ conidia/ml⁻¹, while those of the least pathogenic ranged from 1.6 x 10⁷ to 3.7 x 10⁷ conidia/ml⁻¹. Thaumatotibia leucotreta final instar larvae were exposed to two conidial concentrations, at four different exposure times (12, 48, 72 and 96 hrs) and showed an exposure time-dependant relationship (F=5.43; df=3; P=0.001). At 1 x 10⁷conidia/ml⁻¹ two Beauveria isolates: G Moss R10 and G 14 2 B5 were able to elicit a response in 50% of test insects at 72 hrs (3 days) exposure. Although a limited amount of mycosis was observed in the puparia of both fruit fly species, deferred adult mortality due to mycosis was high. The increased incidence of adult mortality suggests that post emergence mycosis in adult fruit flies may play a more significant role in field suppression than the control of fruit flies at the pupal stage. The increased incidence of pupal mortality, as well as the relatively low concentrations of conidia required to elicit meaningful responses in T. leucotreta pupae may suggest that pre-emergent control of false codling moth will play a more significant role in field suppression than the control of adult life stages using indigenous isolates of entomopathogenic fungi. Various entomopathogenic fungal application techniques targeted at key insect pests within integrated pest management (IPM) systems of citrus are discussed.
- Full Text:
- Date Issued: 2010
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