Endophytic control of Cosmopolites sordidus and Radopholus similis using Fusarium oxysporum V5w2 in tissue culture banana
Dennis M.W. Ochieno
Thesis committee
Thesis supervisors Prof. dr. Marcel Dicke Professor of Entomology Wageningen University
Prof. dr. ir. Arnold van Huis Personal Chair at the Laboratory of Entomology Wageningen University
Thesis co-supervisor Dr. Thomas Dubois Biocontrol Specialist International Institute of Tropical Agriculture
Other members Prof. dr. TWM Kuijper, Wageningen University Prof. dr. RA Sikora, University of Bonn, Germany Dr. JM Raaijmakers, Wageningen University Dr. MNEJ Smit, IPM specialist, (private consultant)
This research was conducted under the auspices of the C. T. de Wit Graduate School of Production Ecology and Resource Conservation Endophytic control of Cosmopolites sordidus and Radopholus similis using Fusarium oxysporum V5w2 in tissue culture banana
Dennis M.W. Ochieno
Thesis submitted in partial fulfilment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. dr. M.J. Kropff in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Monday 1 November 2010 at 11 a.m. in the Aula
Dennis M.W. Ochieno
Endophytic control of Cosmopolites sordidus and Radopholus similis using Fusarium oxysporum V5w2 in tissue culture banana
Thesis, Wageningen University, Wageningen, NL (2010) With references, with summaries in Dutch and English
ISBN 978-90-8585-637-5 Table of contents
Acknowledgements vii Abstract ix Chapter 1 The application of endophytes for the control of pests and diseases in crops and in particular banana 1
Chapter 2 Outline of this thesis 21
Chapter 3 Effects of endophytic Fusarium oxysporum V5w2 as influenced by host nutrition, and the artificial endophyte Beauveria bassiana G41, on the banana weevil Cosmopolites sordidus in tissue culture plants 33
Chapter 4 Comparative studies on effects of NPK nutrient deficiencies and soil sterility on banana plants and Radopholus similis infection 59
Chapter 5 Effects of soil sterilization on interactions between endophytic Fusarium oxysporum V5w2 and the root burrowing nematode Radopholus similis in tissue culture banana plants 87
Chapter 6 Interactions between Radopholus similis and Fusarium oxysporum V5w2 in tissue culture banana plants under nitrogen-starvation and total nutrient starvation as affected by soil sterility 105
Chapter 7 Effects of endophytic Fusarium oxysporum V5w2 on the root burrowing nematode Radopholus similis in
v Table of contents
tissue culture banana plants under phosphorus and potassium deficiencies as affected by soil sterility 125
Chapter 8 Effects of endophytic non-pathogenic Fusarium oxysporum V5w2 and mulching on field-grown tissue culture banana plants and root infection by Radopholus similis 145
Chapter 9 Summarizing discussion 161 References 177 Summary 195 Samenvatting 199 Personal history 205 PE&RC Education statement 207 Funding Acknowledgement 209
vi Acknowledgements
I thank Prof. Arnold van Huis, Prof. Marcel Dicke (WUR), Dr. Thomas Dubois, Dr. Piet van Asten and Dr. Danny Coyne (IITA) for their strong commitment in nurturing me into the world of science. I acknowledge Prof. Joop van Lenteren (WUR), Dr. Cliff Gold (formally of IITA), Dr. Ellie Osir, Dr. Fritz Schulthess (formally of ICIPE), Prof. Micheni Ntiba and Prof. Lucy Irungu (University of Nairobi) for their efforts that led to my recruitment into this great study opportunity. I recognize the technical support that was provided by Abubakar Ezale, Agusi Franco, Arthur Wasukira, Cissy Nabulime, Dennis Nteza, Elvis Mbiru, Fred Kato, Fred Kimuli, Jane Luyiga, Jhamna Castillo, John Kibalama, Joshua Okonya, Juliana Nakintu, Juliet Akello, Margaret Nakawunde, Moses Kibirango, Moses Nyine, Patrick Emudong, Patrick Mayanja, Peter Semakula, Phillip Abidrabo, Ronald Kateriga, Rose Khainza, Stephen Segawa, Sinnia Kapindu, Stephen Ssebaggala, Markstuart Kibirango and Victoria Naluyange among other staff at IITA in Uganda. Fellow students and staff at the Laboratory of Entomology (WUR) provided comments and technical support that greatly improved this thesis. Thanks to Joost van Itterbeeck for the Dutch translation of the summary. I acknowledge the Kenyan and other student associations for creating social and academic occasions. I appreciate the efforts of Judith Naliaka Barasa, Charles Wekulo Barasa, Ashton Okiya, the Baziras, Solomy Kiwanuka, Dr. Edward Muge and Everlyne Kirwa in creating conducive work environment and neighbourhood during my PhD studies. Your prayers and advice played a great role towards the success of my studies.
vii Acknowledgements
To my dad Jackson J. Ochieno, mum Elizabeth N. Ochieno, my son Alvin Jonah Ochieno, all family members and friends, you gave me extra attention during my long absence from home while pursuing studies. Dad and Mum, you sacrificed a lot to ensure that I succeed in this challenging academic journey.
Thanks a lot!
viii Abstract
Banana plants are being inoculated with Fusarium oxysporum V5w2 and Beauveria bassiana G41 for endophytic control of pests. The effects of F. oxysporum V5w2 and B. bassiana G41, soil sterility, fertilizer, and mulching, on Cosmopolites sordidus and Radopholus similis in banana plants, are investigated. Cosmopolites sordidus has low preference for plants inoculated with the two endophytes; corm damage is low on F. oxysporum V5w2-treated plants. High root damage and growth suppression are evident in R. similis-treated plants. Under N-deficiency, R. similis-treated plants are larger than those without the nematode. Compared to plants treated with complete nutrient solution (CNS), those under P-deficiency have higher root damage, but lower under K-deficiency, and not different under N-deficiency and when only water was applied. Plants under CNS have lower R. similis density than those under N-deficiency and those treated with water only. Also, C. sordidus larvae from plants under CNS are smaller than those given only water. Under N-deficiency and supply of only water, potted plants in non-sterile soil are smaller than those from sterile soil, but are larger under CNS, P or K-deficiencies when N is present. Radopholus similis densities are lower in roots from non-sterile soil, compared to those from sterile soil. Mulched plants are larger with bigger bunches than those without mulch, but are more prone to toppling when R. similis is present. Plants treated with F. oxysporum V5w2 have lower R. similis density under N-deficiency but higher under P-deficiency, and are smaller in size under K- deficiency, than endophyte-free ones. Fusarium oxysporum V5w2-treated plants are small and take short time to harvest. With mulch, R. similis- induced toppling is less in F. oxysporum V5w2-treated plants, possibly due
ix Abstract to their smaller size during growth. In conclusion, data on the effect of nutrients, soil microorganisms and mulching do not support the transfer of F. oxysporum V5w2-treated banana plants to farmers, because the plants suffer from reduced performance. Understanding endophytic mechanisms of action and establishing successful inoculation is necessary for drawing a final valid conclusion.
x Chapter 1
The application of endophytes for the control of pests and diseases in crops and in particular banana
Endophytes growing from root pieces in media
Dennis M.W. Ochieno · Marcel Dicke · Thomas Dubois ·
Arnold van Huis
Chapter 1
Abstract Endophytes have become important for plant growth promotion. The technology has been made ready for crops like banana that are seriously constrained by pests and diseases. The current chapter reviews the literature on endophytes with emphasis on the application of non-pathogenic Fusarium oxysporum for the control of banana pests and diseases.
A toppled banana plant
2 The application of endophytes for the control of pests and…………..……..
Pest problems facing banana production in Africa Banana and plantain are grown in over 100 tropical and subtropical countries worldwide (Sharrock and Frison 1999). In Africa, especially in the eastern and central regions of the continent, highland cooking banana cultivars (Musa spp., genome group AAA-EA) are a staple food crop for the livelihood of many people (Pillay et al. 2001). Uganda has been the leading African banana producer, with an annual production of 9.8 million tonnes (Sharrock and Frison 1999), and a consumption rate of 373 kg per capita per year (Smithson et al. 2001). In Rwanda, the annual banana production is 2 million tonnes (39% of total raw food production, with a consumption rate of 197 kg per capita per year) (Nsabimana and van Staden 2007).
a b
Fig. 1.1. The banana weevil Cosmopolites sordidus with eggs (a), and the root
burrowing nematode Radopholus similis coiled inside a root cell (b). Corm damage by the weevil results in snapping while root burrowing by the nematode causes toppling of plants. Images byprovided provided by Moses Nyine and Phillip Abidrabo, respectively.
3 Chapter 1
However, banana production in Africa is threatened by a wide range of pests and diseases. The banana weevil, Cosmopolites sordidus (Coleoptera: Curculionidae) (Figure 1.1) is among the most damaging insect pests that feed on the corms resulting in snapping of plants (Gold et al. 2001). The root burrowing nematode, Radopholus similis (Tylenchida: Pratylenchidae) (Figure 1.1), is among the most damaging species of plant parasitic nematodes worldwide that had been given quarantine status (O'Bannon 1977; EPPO/CABI 1997; Talwana et al. 2003). Other key damaging tylenchid nematode pests include Helicotylenchus multicinctus (Hoplolaimidae), Pratylenchus goodeyi, P. coffeae (Pratylenchidae) and Meloidogyne spp. (Heteroderidae) (Sarah 1989; Speijer and De Waele 2001). Diseases affecting banana plants in Africa include Fusarium wilt (caused by Fusarium oxysporum f. sp. cubense), banana bacterial wilt (caused by Xanthomonas campestris pv. musacearum), black sigatoka (caused by Mycosphaerella musicola) and banana streak virus (Tushemereirwe et al. 2003, 2004). There have also been outbreaks of banana bunchy top viral disease that are a major threat to banana production in Africa (Kenyon et al. 1997; Kumar et al. 2009). All these problems are further complicated by abiotic stresses, such as low soil fertility and drought (Gold et al. 2000; Smithson et al. 2001). Banana has attracted special political support due to the importance of the crop for stability of nations. For example, the Presidential Initiative for Banana Industrial Development (PIBID) by the Government of the Republic of Uganda (PIBID 2008). The success of such initiatives would depend on efforts towards managing pests, diseases and abiotic stresses affecting banana production.
4 The application of endophytes for the control of pests and…………..……..
New technologies are being developed for the management of the problems facing banana production. Of great importance, the concept of endophyte-enhanced banana technology is gaining popularity as a major option in the management of banana pests. This concept emerged following reports that pest-resistant crops contained high levels of endophytes, while pest-susceptible ones had low levels (Ahmad et al. 1986). Various reports have linked resistance to pests by endophyte-containing plants to mechanisms that include toxin production, parasitism, induced resistance, repellence etc. (Breen 1994; Daisy et al. 2002; Sikora et al. 2008). Decrease in pest resistance in crops has been associated with the removal of endophytes from plants (Pedersen et al. 1988; Sikora et al. 2000; Pocasangre 2006). This may be through selective breeding and inoculation of crops with non-toxic endophyte strains (Woodfield and Easton 2004), their deliberate removal from plant tissues (Saiga et al. 2003), and non-target effects of chemicals such as fungicides (Dernoeden et al. 1990). The hope of saving Africa from food shortages seemed to have been ignited by tissue culture technology, which made it possible to mass produce clean banana planting material. However, there have been reports that tissue culture plants are highly susceptible to the same pests they are freed of, as the plants lack endophytes.
What are endophytes and how are they classified? Endophytes are heterotrophic microorganisms that live inside plants primarily for nutrition, protection and reproduction. It is important to note that definitions about endophytes do not draw a clear line between microbes
5 Chapter 1 that are beneficial and those that are pathogenic to crops (Carroll 1988; Azevedo et al. 2000; Backman and Sikora 2008). There is still no solid system of classifying endophytes owing to their complex biological diversity. However, the term ‘endophyte’ is on its own a classification that distinguishes the organisms by the fact that they are endotrophic in plants. Various bases of classifying endophytes are encountered in a wide range of texts. Understanding such bases would help clarify the endophyte concept and the applications of such microbes.
Infected parts and host plants The term ‘endophyte’ distinguishes microbes that invade plant tissues from those that are external. Harish et al. (2008) classified Pseudomonas spp. into plant growth promoting endophytic (PGPE) or rhizosphere (PGPR) bacteria depending on whether they invaded plants or not. However, some endophytic strains of F. oxysporum (A1, V5w2, Fo162 and H2O) can grow in the soil when plants are absent (Vu et al. 2004). Endophytes have been classified based on the part or tissue of the host plant they occupy. Fungi that infect roots such as Fusarium spp. and Glomus spp. are ‘root endophytes’ (Wilberforce et al. 2003; Karandashov et al. 2004). These include ‘root-invading’ microbes that enter into plant tissues from the rhizosphere (Skipp and Christensen 1989), as opposed to those that invade the stems and leaves (foliar endophytes) (Wilson 1993). Some authors seem to have limited the use of the term ‘endophyte’ to microbes that invade the stems and leaves while using the term ‘mycorrhiza’ to refer to those that colonize roots (Wilson 1993).
6 The application of endophytes for the control of pests and…………..……..
The term ‘vascular endophyte’ has been used to refer to microbes such as Xanthomonas campestris pv. campestris that colonizes the vascular system of cabbage (Dane and Shaw 1996). In this respect, pathogenic strains of F. oxysporum that invade the xylem (Olivain and Alabouvette 1999; Recorbet et al. 2003), may be considered to be vascular endophytes, while the non-pathogenic strains may not be vascular endophytes, as they are limited to superficial tissues by plant defences (Olivain and Alabouvette 1999; Recorbet et al. 2003). Infection of banana plants by pathogenic F. oxysporum is always through injured roots, especially via deep wounds that expose the xylem, and there is no evidence of the fungus having an ability to attack living cells of the main root (Raut and Ranade 2004). However, non- pathogenic Fusarium spp. endophytes may invade the vascular system in some special cases. For example, non-pathogenic F. oxysporum V5w2 grew in banana xylem when the roots were broken before inoculation, but only colonized superficial tissues when roots were left intact (Paparu et al. 2009a). Some endophytes have also been classified based on their specialization to host plant groups. Ericoid endophytes such as Hymenoscyphus ericae are common in plants belonging to the family Ericaceae (Read 1996). Neotyphodium spp. are referred to as ‘grass endophytes’ due to their specialization on poaceous hosts (Leuchtmann 1992). In F. oxysporum, plant pathogenic strains cause wilt disease and are grouped into formae speciales (f. sp.) based on their host range, while some are further subdivided into races (Gordon and Martyn 1997). For example, the banana pathogen F. oxysporum f. sp. cubense is divided into four races
7 Chapter 1 that vary in pathogenicity between host plant cultivars (race 1, Gros Michel; race 2, Bluggoe; and race 4, Cavendish; race 3 affects Heliconia species and is only mildly pathogenic to banana) (Ploetz 1990; Bentley et al. 1995).
Taxonomy and structure The taxonomic base of classification has placed most endophytes into two super kingdoms Prokaryotae and Eukaryotae, which include the kingdoms Fungi (fungal endophytes) and Monera (bacterial endophytes), respectively (Ingram 2002). For instance, Fusarium spp. endophytes have been classified into Fungi (kingdom), Ascomycota (phylum), Sordariomycetes (class), Hypocreales (order), Nectriaceae (family) and Fusarium (genus) (Michielse and Rep 2009). However, controversy in classification of the genus Fusarium into species has led to various systems with different numbers of species (Nelson et al. 1994). Endophytic organisms may form characteristic structures by their own cells or in tissues of host plants. Such features have been used to classify endophytes. The bacterial endophytes are prokaryotic as they lack nuclear membranes (Kamoun et al. 1998; Vellai and Vida 1999), as opposed to eukaryotic endophytes that have nuclear membranes (Kupper et al. 2009). Most fungal endophytes, such as F. oxysporum V5w2 (Paparu et al. 2009a), are composed of threadlike structures known as hyphae that form mycelial masses, which earn them the name ‘mycelial endophytes’ (Narisawa et al. 2003). Such structures enable them to grow and acquire resources from host plants and the rhizosphere simultaneously. Vesicular arbuscular mycorrhizal fungi (AMF) e.g. Glomus spp. penetrate plant cortical cell walls and form
8 The application of endophytes for the control of pests and…………..…….. characteristic, densely branched, arbuscules (Guttenberger 2000). Mycelia are also found in the bacterial endophytes known as actinomycetes e.g. Streptomyces spp. (Taechowisan et al. 2003). However, bacterial endophytes like Pseudomonas fluorescens PICF7 exist in single cells that form colonies (Prieto and Blanco 2008). Root nodule bacteria induce the formation of nodules in their host tissues (O'Hara and Glenn 1994).
Genetic Molecular phylogenetic classification of endophytes involves the analysis of nucleic acids and proteins in studying the evolutionary relationships of endophytes. For example, through restriction fragment length polymorphism (RFLP) loci analysis, 72 RFLP haplotypes of F. oxysporum f. sp. cubense infecting banana have been identified (Koenig et al. 1997). A molecular phylogenetic relationship of Epichloë typhina and other clavicipitaceous endophytes, which also shows that endophytes may coevolve with their hosts, was reported by Schardl et al. (1991). Endophytes whose genes have been manipulated by genetic engineering are classified as ‘genetically modified endophytes’ as opposed to the ‘wild-types’ that retain their natural genomes (Murray et al. 1992; Gullino and Migheli 1999). For instance, non-pathogenic F. oxysporum V5w2 was transformed with the green (GFP) and red fluorescent protein (DsRed) genes that facilitated its observation in banana root xylem since the wild-type could not be observed (Paparu et al. 2009a).
9 Chapter 1
Nutrition All endophytes are heterotrophs (organotrophs), since they acquire carbon in the form of organic compounds, unlike green plants that utilize CO2 (Pace 1997). They are also sub-classified as chemoorganotrophs since they utilize organic substances for energy, which contrasts their host plants that use light energy in photosynthesis (Broda and Peschek 1984). Depending on whether they gain nourishment from dead or living materials, endophytes can be classified as saprophytes, necrotrophs, or biotrophs (Varma et al. 1999). Saprophytic stages of endophytes such as non-pathogenic F. oxysporum are free-living in the rhizosphere (Nel et al. 2006), while biotrophic phases establish inside living hosts (Bacon et al. 2001; Salerno et al. 2004). Necrotrophic endophytes on the other hand feed on dead tissues (Varma et al. 1999; Idnurm and Howlett 2001). These include strains of non-pathogenic F. oxysporum that are associated with necrotic root tissues (Speijer and Sikora 1993; Sikora et al. 2008). Endophytes can be classified based on their ability to utilize or grow under specific nutrient stresses. For instance, nitrate-nonutilizing (nit) (or chlorate resistant) mutants of F. oxysporum can be generated by growing the wild-types in media containing potassium chlorate (Puhalla 1985; Katan and Katan 1988). The ‘nit’ mutants are classified into three categories that reflect mutations at a nitrate reductase structural locus (nit1), a nitrate- assimilation pathway-specific regulatory locus (nit3), and loci that affect the assembly of a molybdenum-containing co-factor necessary for nitrate reductase activity (NitM) (Zamani et al. 2004). A chlorate-resistant F. oxysporum V5w2 (nit3) is currently under investigation for the management
10 The application of endophytes for the control of pests and…………..…….. of the nematode R. similis in banana plants (Paparu et al. 2009a,b). Based on the source of nitrogen, symbiotic microbes such as Rhizobium spp., Azoarcus spp., Herbaspirillum spp. and Acetobacter diazotrophicus, which can utilize nitrogen gas, are classified as diazotrophic endophytes (Christiansen 1998; Hurek and Hurek 1998).
Reproduction Based on the mode of reproduction, endophytes can be grouped as asexual or sexual (Brem and Leuchtmann 2001). For example, the Epichloë endophytes have been divided into the genus Epichloë that reproduces sexually and the genus Neotyphodium (formally Acremonium) that only reproduces asexually (Moon et al. 1999; Leuchtmann et al. 2000; Schardl and Craven 2003). Sexual reproduction does not exist in F. oxysporum endophytes and they rely on asexual propagation (Gordon and Martyn 1997). Another system of classifying endophytes, known as vegetative- compatibility grouping (VCG), is based on the ability of mycelia from two strains to anastomose and form heterokaryons (Puhalla 1985; Katan and Katan 1988). Paparu et al. (2008) applied the VCG technique in detecting non-pathogenic F. oxysporum strains V5w2 and III4w1 to verify their persistence in inoculated banana roots.
Transmission Endophytes are also classified based on their mode of transmission in host populations. Vertically-transmitted endophytes are passed directly from host plants to their offspring (Saikkonen et al. 2002). Those vertically
11 Chapter 1 transmitted via the host seeds are referred to as seed-transmitted endophytes (Dongyi and Kelemu 2004), for instance, F. oxysporum f. sp. vasinfectum race 4 in cotton (Bennett et al. 2008). On the other hand, horizontally- transmitted endophytes are contagiously transferred between different individuals in a population (Saikkonen et al. 2002). Such endophytes may be multiplied through vegetative propagules e.g. banana suckers and tissue culture plantlets (Thomas et al. 2008), or transmitted via spores hence spore- transmitted endophytes (Faeth and Fagan 2002). The transmission of microbes between plants has been used to classify undesired endophytes as contaminants (Barker et al. 2005; Paparu et al. 2006a).
Pathology and toxicology In phytopathology, endophytes that cause disease in infected plants are phytopathogens. The term ‘non-pathogenic’ has been used to describe (strains of) endophyte species that do not harm their hosts, while putting into consideration that closely related pathogenic variants do exist. For example, non-pathogenic F. oxysporum V5w2 (Paparu et al. 2009a) are closely related to pathogenic F. oxysporum f. sp. cubense strains that cause banana wilts (Koenig et al. 1997). In addition, latent pathogens and those of very low virulence may be considered as (non-pathogenic) endophytes (Carroll 1988; Photita et al. 2004). The terms ‘symptomatic’ and ‘asymptomatic (symptomless)’ classify endophytes depending on whether the host plants express infection symptoms (Pinto et al. 2000; Araújo et al. 2002). For instance, a wide range of endophytes especially Fusarium spp. were obtained from asymptomatic cord roots of the banana cultivar Pisang
12 The application of endophytes for the control of pests and…………..……..
Awak (Musa ABB) (Niere 2001; Sikora et al. 2008). Symptomatic endophytes may sometimes be qualified as asymptomatic when the host plant is resistant (Araújo et al. 2002). In a crop such as banana, young plants do not develop symptoms of infection with the pathogen F. oxysporum f. sp. cubense (Ploetz 1998). Based on the symptoms of infection with F. oxysporum f. sp. cubense, this strain is divided into four races that vary in pathogenicity between host plant cultivars: race 1, Gros Michel; race 2, Bluggoe; race 3 affects Heliconia species and is only mildly pathogenic to banana; and race 4, Cavendish banana (Ploetz 1990; Bentley et al. 1995). Although defence reactions of plants such as banana are induced when invaded by pathogenic endophytes like F. oxysporum f. sp. cubense (Thangavelu et al. 2003), similar effects also occur when they are infected with non-pathogenic strains e.g. F. oxysporum V5w2 (Paparu et al. 2007). Pathogenic strains of F. oxysporum have been labelled ‘root-rot fungi’ owing to their contribution towards root necrosis (Benhamou et al. 1996). Some non-pathogenic strains of F. oxysporum also play a role in root rots (Speijer and Sikora 1993). In zoopathology, microbes can colonize plant tissues and negatively affect pests or livestock through direct or indirect mechanisms. In pest management, those that colonize plant tissues and infect insects are called entomopathogenic endophytes e.g. Beauveria bassiana and Clonostachys rosea (Vega et al. 2008). The entomopathogenic characteristic may be through direct parasitism such as mycosis by B. bassiana G41 on the banana weevil Cosmopolites sordidus (Akello et al. 2007,2008), or indirectly through toxin production and induced resistance in the plant, as caused by
13 Chapter 1
F. oxysporum against the nematode R. similis in banana plants (Athman et al. 2006, 2007; Sikora et al. 2008). In addition to the above bases of classifying endophytes, other systems have emerged. Endophytes have been classified into different strains or isolates with code names that are determined by collectors and storage systems (e.g. Forsyth et al. 2006). This may be the most popular system among endophytologists, although it lacks common rules. It may be difficult to get the true meaning of the code names unless explained. For instance, five isolates of non-pathogenic F. oxysporum being tested for the control of R. similis in banana include strain Emb2.4o, Eny1.31i, Eny7.11o, V4w5 and V5w2 (Paparu et al. 2009a). The meanings of such code names could not be verified. Microbes that invade internal plant tissues by stochastic events have been classified as ‘passenger endophytes’, while those with adaptive traits enabling them to strictly live in association with the plant as ‘true endophytes’ (Almeida et al. 2009). The term ‘artificial endophyte’ has been applied to microbes that naturally do not occur in plant tissues as their ecological niche, but may be detected in plant tissues after inoculation e.g. the entomopathogenic fungus B. bassiana G41 (Akello et al. 2007).
In summary, all the classifications of endophytes are based on the fact that the microbes exist inside plant tissues. Therefore, any microbe that may be detected in plant tissues qualifies as an endophyte regardless of its effects on the host plant. This includes naturally occurring microbes in plant tissues (Clay 1988; Sikora et al. 2008), those that may be observed in plant
14 The application of endophytes for the control of pests and…………..…….. organs (Paparu et al. 2006a, 2009a), those that may be extracted from surface-sterilized plant tissues (Sardi et al. 1992; Cao et al. 2005), those that may be re-isolated following inoculation (Paparu et al. 2006a; Akello et al. 2007), and the ones considered to be contaminants (Barker et al. 2005; Paparu et al. 2009b).
The tissue culture option in the control of pests and diseases of banana The use of clean planting materials, which are free of pests and diseases, as well as being resistant to biotic and abiotic stresses, is a fundamental measure for banana production. In addition to these qualities, the plants need to be high yielding and acceptable to consumers. Clean banana planting material can be obtained by hot-water treatment of pared corms and by tissue culture (Bridge 1996).
a b c
Fig. 1.2. A tissue culture banana plantlet in a test tube containing rooting medium before being transferred into nutrient solution (a), a hydroponically grown banana plantlet in nutrient solution contained in a plastic cup maintained inside a humidity chamber before being grown in hardening bags (b), and the plants grown in hardening bags containing sterilized soil in the screenhouse before being transferred to the field (c).
15 Chapter 1
Tissue culture has become a popular biotechnical tool in the face of high demand for clean planting material (Krikorian and Cronauer 1984; Vuylsteke 1998) (Figure 1.2), triggered by food shortages due to pests and abiotic stresses (Jain 2001; Gold et al. 2002). The production of clean planting material for banana by tissue culture starts with the selection of suckers or other tissues from healthy donor plants (Leifert and Cassells 2001), cleaning them of pests and pathogens and multiplying them. This involves stringent aseptic measures on plant material, media and equipment. The process employs chemical detergents (e.g. ethanol and hypochlorite), antibiotics, and heat-sterilization in autoclaves or by flaming (Vuylsteke 1998). During the tissue culture process, there is a consequent loss of beneficial microbes that contribute to plant vigour and performance in the field (Sikora et al. 2000; Schmidt et al. 2004). Tissue culture banana plants have been considered to be susceptible to pests and pathogens due to loss of protective endophytes (Sikora et al. 2000; Lian et al. 2009).
The endophyte solution to the drawback of tissue culture banana in pest control The view that tissue culture banana plants are susceptible to pests in the field has led to the concept of reintroducing protective microbial endophytes (Sikora et al. 2000; Schmidt et al. 2004; Lian et al. 2009). This innovation is referred to as the endophyte-enhanced banana tissue culture technology, which is being developed by various research organizations worldwide including the International Institute of Tropical Agriculture (IITA) in Uganda (Dubois et al. 2004, 2006a; Pocasangre 2006; Figure 1.3). Banana
16 The application of endophytes for the control of pests and…………..…….. plants are obtained by tissue culture techniques, such as the shoot-tip culture method described by Vuylsteke (1998). The plants may be inoculated with protective microbes, obtained from healthy crops, at various stages before being transferred to farmers. Endophyte inocula are prepared depending on the target pest, type of microbe, stage of plant growth, and the desired inoculation method. Inoculum formulations are prepared by methods based on microbial- phytopathological techniques. These include suspensions of microbial cells, spores and hyphae (liquid inocula) (Paparu et al. 2006a; Wang et al. 2007), or microbial cultures in solid substrates (Paparu et al. 2006b). Spore suspensions in sterilized water and cereal bran solid substrates have been used in the preparation of inocula for endophytic F. oxysporum V5w2 and B. bassiana G41 in banana plants (Akello et al. 2007; Paparu et al. 2009b). Plants may be inoculated with liquid suspensions by root dipping of soilless roots (Paparu et al. 2006a), drenching of roots together with rhizosphere soil (Kavino et al. 2007), pipetting the inoculum into the rhizosphere (Lian et al. 2009), and by injecting the plants (Akello et al. 2007). Solid substrates are sprinkled on roots and mixed with soil before planting (Paparu et al. 2006b; Akello et al. 2007). The plants are grown in sterilized soil to enhance root colonization by endophytes, while preventing contamination with other microbes. The success of inoculation with endophytes like chlorate-resistant F. oxysporum V5w2 (nit3) can be assessed by re-isolation from surface- sterilized tissues using selective media (Paparu et al. 2009b), histological techniques (Paparu et al. 2006a), especially with visual enhancements such as transformation using fluorescence genes (Paparu et al. 2009a).
17 Chapter 1
Isolation of naturally-occurring endophytes from healthy plants
Culturing, identification, purification and storage
Laboratory bioassays against Development of cost-effective and target pests efficient inoculation technique
Assuring non-pathogenicity on Screenhouse bioassays of Establishing colonization success both hosts and non-host plants endophyte-enhanced plants
Marking promising endophyte Field experiments of endophyte- Determining mode-of-action and strains enhanced plants endophyte persistence
Identification of private partnership
On-farm experiments of endophyte-enhanced plants
Fig. 1.3. Overview of the research protocol used for developing endophyte-enhanced tissue culture banana plants (Dubois et al. 2006a,b).
As a routine procedure, endophyte-enhanced plants are tested for protection against pests and diseases. This involves assessment of levels of pest damage on plants (Figure 1.4). In the case of nematodes such as R. similis, there is application of protocols such as those of Speijer and Gold (1996), Speijer and De Waele (1997) and Brooks (2004). This has been applied for F. oxysporum V5w2 against R. similis in banana plants (Athman et al. 2007; Paparu et al. 2009b). For the banana weevil C. sordidus, corm damage assessment may be undertaken based on techniques like those of
18 The application of endophytes for the control of pests and…………..……..
Ortiz et al. (1995) and Gold et al. (2004a, 2005). However, a wide range of damage assessment methods for corm damage by C. sordidus exist and there are no agreed upon assessment protocols (Gold et al. 2004a). The assessment methods also include evaluation of the effects of endophytes on plants health, which include plant growth parameters (Akello et al. 2007; Paparu et al. 2009b). Direct and indirect effects of endophytes on the pests are also assessed to clarify the mechanisms of action (Sikora et al. 2008). The endophyte-enhanced banana technology has been ready for farmers’ fields (Pocasangre 2006; Dubois et al. 2006b). In the technology, F. oxysporum V5w2 is among endophytic fungi that have shown biological control potential against C. sordidus and R. similis (Athman et al. 2007; Paparu et al. 2009b). Information on the effects of ecological factors and crop management practices on endophyte-enhanced banana plants would facilitate transfer of the technology to farmers.
a b
Fig. 1.4. Cross section of a healthy banana corm and one tunneled by Cosmopolites sordidus (a), and
healthy roots and those with lesions caused by Radopholus similis (b). Images by Moses Nyine.
Acknowledgements We acknowledge Dr. Danny Coyne for reviewing this chapter.
19
Chapter 2
Outline of this thesis
Inspection of plants in the screenhouse (Arnold and Dennis)
Dennis M.W. Ochieno Chapter 2
Background information The production of East African highland cooking banana in Africa has been hindered by the banana weevil Cosmopolites sordidus and the nematode Radopholus similis (Price 1994; Gold et al. 2001). The use of pest-free planting material for banana is being encouraged to minimize plant damage by these pests. Tissue culture is a biotechnical tool increasingly promoted for production of pest-free banana planting material (Vuylsteke 1998; Strosse et al. 2006). However, there have been concerns that tissue culture eliminates protective microbes, which makes banana plants vulnerable to re-infestation by pests in the field (Sikora et al. 2000). Such concerns have led to research into endophytic microbes for the control of plant-parasitic nematodes, especially endophytic strains of Fusarium oxysporum that are non- pathogenic to crops (Sikora et al. 2008). The beneficial effects of endophytic F. oxysporum strains on plant performance and induced pest resistance has been documented in a series of researcher-controlled pot trials (Vu et al. 2006; Athman et al. 2007; Paparu et al. 2009b; Sikora et al. 2008). Various research organizations worldwide including the International Institute of Tropical Agriculture (IITA) have been carrying out research on the introduction of endophytic microbes into tissue culture banana plants to offer protection against pests (Dubois et al. 2006a,b). Endophytic F. oxysporum strain V5w2 has been considered to have biological control potential against R. similis (Athman et al. 2007). The entomopathogenic fungus Beauveria bassiana strain G41 is also being tested as an artificial endophyte in banana plants against C. sordidus (Akello
22 Outline of this thesis et al. 2007). The technology of enhancing tissue culture banana plants with microbial endophytes has been made available for farmers’ fields (Pocasangre 2006; Dubois et al. 2006b). However, it is not sufficiently clear how the results from controlled experiments will relate to on-farm conditions where other biotic and abiotic factors affect plant performance, thereby possibly affecting the endophyte efficacy to combat pests.
About the thesis IITA among other research organizations aims at providing farmers with endophyte-enhanced tissue culture banana plants. To achieve this goal, information is required on the effects of soil biotic and abiotic factors, as well as crop management practices, on the performance of the endophytes against banana pests. This has been the topic of the current PhD research. Fusarium oxysporum V5w2 was targeted for this research as it had been identified as the most promising strain against R. similis among a collection of endophytes at IITA in Uganda (Paparu et al. 2006a; Athman et al. 2007). The aim of the research described in this PhD thesis was to provide in-depth information on effectiveness of endophytic F. oxysporum V5w2 and B. bassiana G41 in the control of banana pests before transferring the endophyte technology to farmers. Through laboratory, screenhouse and field experiments, the effects of soil biotic factors (microorganisms), abiotic factors (nutrients), and crop management practices (mulching) on pest control in F. oxysporum V5w2-inoculated banana plants have been investigated. Laboratory experiments have been employed to investigate the behaviour of C. sordidus towards banana plants inoculated with B. bassiana
23 Chapter 2
G41. If endophyte-treated plants are unattractive and resistant to the pests, this would lower the fitness of C. sordidus and R. similis.
Objectives of the studies in this thesis The main objective of the research described in this thesis was to elucidate the effects of major plant nutrients, soil microorganisms and mulching on the potential of endophytic F. oxysporum V5w2 in the control of banana pests. Some concepts and techniques that had already been developed by other researchers in the endophyte-enhanced banana tissue culture technology have been applied (see Dubois et al. 2006b; Figure 1.3). The study had three specific objectives: (1) to evaluate the effects of F. oxysporum V5w2 and B. bassiana G41 on C. sordidus in tissue culture banana plants; (2) to evaluate the effects of nitrogen (N), phosphorus (P), potassium (K) and soil microorganisms on the control potential of endophytic F. oxysporum V5w2 against R. similis; (3) to evaluate the effects of mulching on the control potential of F. oxysporum V5w2 against R. similis. These objectives are addressed in the experimental research described in chapters 3-8.
Effects of Fusarium oxysporum V5w2 and Beauveria bassiana G41 on Cosmopolites sordidus in tissue culture banana plants Chapter 3 addresses the effects of endophytic F. oxysporum V5w2 and B. bassiana G41 on the banana weevil, C. sordidus, in banana plants. This was based on the hypothesis that, apart from having direct effects such as parasitism on the weevils or their eggs (Dubois et al. 2006a; Akello et al.
24 Outline of this thesis
2007), the two microbes when used as endophytes may cause physiological changes in the host plant that may affect the insects indirectly. This objective has been realized through laboratory, screenhouse and field experiments with potted banana plants, which were conducted between July 2007 and November 2008. Experiments with F. oxysporum V5w2 have utilized plants that were either fertilizer-treated or not to assess the effects of nutrition and endophyte inoculation on host preference and suitability for C. sordidus. The effects of F. oxysporum V5w2 and fertilizer application on the suitability of banana plants to C. sordidus, has been addressed by assessing larval growth and the feeding damage they cause in their hosts. The preference behaviour of C. sordidus towards such hosts has been studied by utilizing intact plants and their tissues in olfactometers under laboratory conditions, or by using intact potted plants in the field. The effect of B. bassiana G41 on host choice by C. sordidus has been addressed through olfactometer experiments with tissues from potted banana plants that had been inoculated with the fungus or left uninoculated.
Effects of N, P, K and soil microorganisms on the control potential of Fusarium oxysporum V5w2 against Radopholus similis The studies addressing this topic are presented in chapters 4-7. These studies involved three screenhouse experiments with the code names Biotic- N, Biotic-P and Biotic-K (Table 2.1a,b). The aim for establishing these experiments was to investigate the effect of nutrient deficiencies, soil microorganisms, the nematode R. similis and the endophyte F. oxysporum
25 Chapter 2
V5w2 on the banana plant. The nutrient deficiencies were addressed through nutrient omission treatments (Amberger 2006), while the effects of soil microorganisms were addressed by comparisons between sterile and non- sterile soil (Troelstra et al. 2001). Each of the three experiments (Biotic-N with three nutrient treatments; Biotic-P and Biotic-K each with two nutrient treatments – see Table 2.1a, b) studied the effect of the three factors: the nematode R. similis (with and without), the endophyte F. oxysporum V5w2 (with and without), and the effect of soil microorganisms (by using sterile and non-sterile loamy soil). This means a 3×2×2×2 factorial experiment (24 treatments) for Biotic- N, and a 2×2×2×2 factorial experiment (16 treatments) in the case of Biotic- P or Biotic-K. Each treatment consisted of 15 potted banana plants. The experiments focused on N, P and K as these are the major nutrient deficiencies in soil that greatly limit East African highland cooking banana production (van Asten et al. 2004). Comparison of sterile and non-sterile soil treatments was used to investigate the effect of soil microorganisms as they interact with endophytes in the rhizosphere and when they invade roots. Such interactions had not been addressed in previous studies. Data were collected on parameters related to nematode multiplication in tissues, root damage and plant growth. The effect of soil microorganisms has been elaborated by extraction and identification of root-invading fungi from banana plants, which were tested in vitro for inhibitive interactions with F. oxysporum V5w2. In each experiment there were numerous statistically significant interactions between the nutrient, nematode, endophyte and soil sterility
26 Outline of this thesis
(microorganisms) treatments on R. similis infection and plant growth parameters. This made it difficult to clearly report the complex results in a single chapter. For instance, the effects and densities of R. similis in plants varied between nutrient treatments. The effects of F. oxysporum V5w2 on R. similis also varied ranging from no effect, suppression or enhancement of the pest. Such variations in R. similis density have been reported by Athman et al. (2007), and also occur in the growth of host plants that are inoculated with endophytes (Paparu et al. 2009b). It was a challenge to identify the nutrient treatments under which F. oxysporum V5w2 provided the best or worst control of R. similis. Such inconsistencies refocused the work from the subject of biological control towards understanding the outcomes of plant-microbe-nematode interactions with abiotic (i.e. nutrients) and biotic factors. The effects of F. oxysporum V5w2 on R. similis would therefore be best addressed under the specific nutritional conditions. Also, the variation in R. similis infection symptoms due to nutrient treatments and soil sterility would be clearly addressed in plants that were not inoculated with the endophyte. Therefore, retrospectively, it was decided to split the data for the three experiments into specific entities and using them in particular chapters. It was considered that this would address the effects of the treatments better while capturing details instead of using the experiments as chapters. The way this was done is presented in Table 2.1a. After initial analysis of data in experiment Biotic-N, then experiment Biotic-P and Biotic-K, the complete nutrient solution (CNS) treatment yielded results that were consistent between the three experiments. In order to avoid repetitions,
27 Chapter 2
f
o
l e contro h F. oxysporum by nitrogen (N). Part II nking the endophyte, ents on on t ents . . P-def P-def K-def i o2 b2 R. similis +Sterile soil+CNS’ nutr d Water Water N-def N-def R. similis sms an sms i Biological control of V5w2 asby affected loamy soil sterility within specific plant nutritional conditions Chap. 5 Chap. 6 Chap. 7 Chap. croorgan i m
il R. so f ects o
ff e data h t of d in banana plants plants banana in Separatedata sets, analyzed separately gate i Effects of loamy soil sterility and fertility on symptomsnutrientof by damage and starvation similis 4.2 4.3 4.4 5.1 6.1; 6.2 7.1 7.2 6.2 4.2 4.3 4.4 5.1 6.1;
b2, o2 Biotic-N Biotic-P Biotic-KBiotic-N,P,K Biotic-N Biotic-P Biotic-K * treatment The combination endophyte+ is ‘No PART II: Thesis chapters and sources of data data of sources and chapters Thesis II: PART 4 Chap. 4 nvest i N-def P-def K-def . d at h
. . Water . . . c Biotic-K 3 ments t ments i K-def K-def c
Biotic-P potassium Total nutrient deficiency 4
e 2 ouse exper ouse h -N P-def .
b 1 CNS CNS CNS * CNS CNS CNS . . . e V5w2 in banana plants, and the integration of data into Chapters 4, 5, 6 and 7. Part I presents the the I presents 7. Part 6 4, 5, and Chapters into of data integration the and plants, banana in V5w2 a not treated 0 Nitrogen-deficientsolution phosphorus 1080 720 720 540 360 360 1080 360 each 360 360 360 360 1080 each 720 720 540 360 360 1080 360 15 15 15 15 15 15 15 15 each 15 15 15 15 each 15 15 15 15 15 15 15 Experiments (lasting 100 days) 100 (lasting Experiments 8 8 each 8 8 8 8 3 3 12 24 16 16 each3 3 3 3 9 3 3 3 Biotic b 3 ree screen h V5w2 n t i nitrogen nitrogen 2 Sterile CNS Sterile CNS CNS CNS .. . CNS . . . CNS CNS CNS Sterile Potassium-deficient solution d
r Fusarium oxysporum Fusarium treatments l a by i Fusariumoxysporum f
Objective(s)of the chapter Data location in thethesis (Table) N (plants/experiment) N (plants/experiment) n (plants/rep/ trt) N-def P-def K-def . P-def K-def N-def N-def P-def K-def . P-def K-def N-def P-def K-def N-def N-def P-def K-def . P-def K-def N-def P-def K-def N-def P-def K-def N-def Treatments (trt) (rep) Replicates R. similis Factor R.similis f
a. 1 . 2
0 Soil bioticfactors (microorganisms) Complete nutrient solution (adequate nutrition) Radopholus similis
PART I: andExperiments treatments Source Treatments Endophyte Non Nematode Soil N-def Water solution Nutrient . . . CNS . CNS Non-Sterile . Water Water CNS Water Non CNSCNS CNS . . . Sterile CNS oxysporum F. . K-def . CNS . Water Water Water .. CNS . CNS . . . CNS CNS . P-def . . . . . CNS CNS . K-def CNS CNS . . . CNS Non-Sterile K-def N-def Water Water CNS Water CNS CNS CNS .. . CNS P-def . . . P-def . . . CNS N-def . K-def . CNS . Non-Sterile . K-def N-def Water . CNS ...... CNS . . . .P-def P-def . . Sterile CNS Non N-def . Water K-def CNS . CNS . K-def N-def Water . . . . P-def P-def . . N-def . Water Non-Sterile . K-def N-def Water . . . . P-def N-def . Water . Water . . . . Water 1 r a solution Phosphorus-deficient treatments within the three staggered factorial experiments, each with a code name e.g. Biotic-N stands for biotic and factors for stands name Biotic-N e.g. a code with each experiments, factorial staggered three withintreatments the obtainedby be li can combination Awere respectivein treatment chapters. the presents that treatments nutrient considered the nematode and soil treatmentsnutrientwith a solution row for each the same in experimentor chapter. Table Radopholus similis
28 Outline of this thesis
Table 2.1b. Nutrient treatments in three screenhouse experiments Biotic-N, Biotic-P and Biotic-K that investigated the control of Radopholus similis by Fusarium oxysporum V5w2 as affected by soil sterility in the years 2007 and 2008.
Experiment Nutrient treatments Replicates (code names) Complete Nutrient Non (CNSa) omissions (only water) Rep. 1 (May 2007 - Aug 2007), Rep. 2 (Sep 2007 - X* X (without N) X Biotic-N Jan 2008), Rep. 3 (Feb 2008 - May 2008) Rep. 1 (July 2007 - Nov 2007), Rep. 2 (Nov 2007 - Biotic-P X X (without P) Mar 2008), Rep. 3 (May 2008 - Sep 2008) Rep. 1 (July 2007 - Oct 2007), Rep. 2 (Nov 2007 - Biotic-K X X (without K) Feb 2008), Rep. 3 (July 2008 - Oct 2008) aComplete nutrient solution i.e. adequate nutrition (Murashige and Skoog 1962) * ‘X’ means eight treatments: R. similis (yes/no), F. oxysporum V5w2 (Yes/No) and soil microorganisms (sterile and non sterile) in a full factorial design (Details in Table 2.1a). the data of CNS-treated plants from the three experiments were combined to assess the effects of F. oxysporum V5w2 against R. similis under such nutrient conditions. This is presented in chapter 5. Plants that received solutions that were deficient in N, P and K or no nutrients (i.e. only water added) in the respective experiments expressed effects that were unique to each nutrient treatment. Data for plants that received N-deficient solution or water only were analyzed separately for the control of R. similis by F. oxysporum V5w2. These are presented in chapter 6. Separate data analyses for plants that received solutions that were deficient in P and K for the suppressiveness of F. oxysporum V5w2 on R. similis are presented in chapter 7. However, such data formations in chapters 5-7 cannot simultaneously compare the symptoms of nutrient starvation and variations in R. similis infection between the nutrient treatments. Therefore, three data
29 Chapter 2 sets were analyzed for this purpose, by only considering plants that were not inoculated with F. oxysporum V5w2, in experiment Biotic-N, experiment Biotic-P and experiment Biotic-K, respectively. This is presented in chapter 4. Soil sterility treatments were maintained as the effects of soil microorganisms that always exist in the field had not been addressed in previous studies.
Effects of mulching on the biological control potential of Fusarium oxysporum V5w2 against Radopholus similis Chapter 8 aims at clarifying the compatibility of F. oxysporum V5w2 with mulching in the integrated pest management (IPM) for banana. Mulching was chosen as a field management practice as it is important in improving soil quality and mitigating the impact of nematodes (McIntyre et al. 2000). Compatibility between F. oxysporum V5w2 as a biological control agent, with mulching as a cultural practice, would be beneficial for IPM in banana. A full-factorial field experiment (2×2×2) was set up with tissue culture banana plants that were treated with R. similis or not, that were inoculated with F. oxysporum V5w2 or not, on mulched or unmulched plots. This experiment was conducted between October 2006 and February 2009. Data were collected on attributes related to plant infection by F. oxysporum V5w2 and R. similis, root damage, plant growth and banana yields. The chapter also addresses the effects of F. oxysporum V5w2 and mulching on non-target species of plant-parasitic nematodes that occurred naturally in the experimental field.
30 Outline of this thesis
Summarizing discussion and conclusions Chapter 9 presents an analysis on the control of R. similis and C. sordidus using microbial endophytes in tissue culture banana plants. The aim of this chapter is to link up the concepts and results from the other chapters. Findings in this thesis have been discussed in relation to other studies. Knowledge gaps have been identified and recommendations for future studies have been made. In this chapter, I have concluded my work based on the observations that I made on the use F. oxysporum V5w2 and B. bassiana G41 as endophytes for the control of C. sordidus and R. similis in banana plants.
Laboratory discussions (Rose, Dennis and Victoria)
Acknowledgements I thank Prof. Arnold van Huis and Prof. Marcel Dicke for contributing research approaches and editorial comments towards this chapter. Thanks to Dr. Thomas Dubois, Dr. Danny Coyne and Dr. Piet van Asten for reviewing this chapter.
31
Chapter 3
Effects of endophytic Fusarium oxysporum V5w2 as influenced by host nutrition, and the artificial endophyte Beauveria bassiana G41, on the banana weevil Cosmopolites sordidus in tissue culture plants
Preparation of endophyte cultures in a laminar flow hood (Patrick and Nuruh)
Dennis M.W. Ochieno · Jhamna Castillo · Marcel Dicke · Thomas Dubois · Danny Coyne · Arnold van Huis
Chapter 3
Abstract We investigated the effects of endophytic Fusarium oxysporum V5w2, plant nutrition, and Beauveria bassiana G41 on olfactory cues and host suitability of tissue culture plants to the banana weevil Cosmopolites sordidus. In the laboratory, (a) more banana weevils chose for the volatiles from intact control plants than clean air, (b) banana weevils did not discriminate between clean air and volatiles from F. oxysporum V5w2-treated plants, and (c) banana weevils did not discriminate between volatiles from control plants and F. oxysporum V5w2-treated plants. In the field, fewer adult banana weevils were found on F. oxysporum V5w2-inoculated plants than on non-inoculated plants 12 h after insect release. Both in the laboratory and in the field, banana weevils did not discriminate between nutrient-treated plants and those without nutrients. Lower numbers of banana weevils chose sections from B. bassiana G41-inoculated plants than those not inoculated. The amount of corm damage by C. sordidus larvae was lower in F. oxysporum V5w2-inoculated plants than in non-inoculated ones, which resulted in better growth of F. oxysporum V5w2-inoculated plants. Larvae from nutrient-treated plants were less heavy than those that fed on plants that were not treated with nutrients. Based on these results, we conclude that, under sterile soil conditions, inoculating tissue culture banana plants with F. oxysporum V5w2 and B. bassiana G41 may negatively affect host preference of adult C. sordidus, and suppress corm damage by their larvae. Larval C. sordidus grow better on nutrient-limited plants, but soil fertility did not affect host-derived cues for the adults.
34 Effects of endophytic Fusarium oxysporum V5w2 as influenced by host…..
Introduction The banana weevil Cosmopolites sordidus and parasitic nematodes inflict heavy damage on banana plants (Speijer et al. 1993; Price 1994). Corm feeding by C. sordidus results in snapping of banana plants (Gold et al. 2001). Plant damage by pests threatens the livelihoods of many people in Eastern and Central Africa who rely on highland cooking banana as a primary food crop. The management of C. sordidus in banana has been very difficult owing to its cryptic feeding in plant tissues, which makes the pest less susceptible to control measures. Tissue culture is a technique that is currently being employed for rapid production of pest-free banana planting material (Vuylsteke 1998). However, tissue culture banana plants are considered vulnerable to re-infestation by field pests, since protective endophytic microbes are eliminated by the stringent aseptic protocols (Sikora et al. 2000). The vulnerability of tissue culture banana plants to re-infestation by pests has prompted research on the introduction of microbial endophytes to enhance plant protection (Sikora et al. 2000; Dubois et al. 2006a). For example, endophytic Fusarium oxysporum V5w2 has been shown to have suppressive effects against the nematode Radopholus similis in banana roots (Athman et al. 2007). Information on the effects of F. oxysporum towards C. sordidus is currently limited. Niere et al. (2004) suggested that some strains of F. oxysporum could suppress banana plant damage and constrain the growth of C. sordidus larvae. Paparu et al. (2009b) found that F. oxysporum isolates V5w2 and Emb2.4o were suppressive to C. sordidus in banana plants when applied in combination. Suppressiveness of F. oxysporum on C.
35 Chapter 3 sordidus has been linked with antibiosis (Griesbach 2000; Paparu et al. 2009b). The entomopathogenic fungus Beauveria bassiana strain G41 has been inoculated into banana plants as an artificial endophyte for the management of C. sordidus (Akello et al. 2007, 2008). This artificial endophyte expresses its entomopathogenic properties against C. sordidus while inside banana tissues (Akello et al. 2008). There have been suggestions that microbes may influence the production of banana plant odours, causing an altered olfactory response by C. sordidus (Braimah and van Emden 1999). The contribution of plant nutrition to endophytic control of C. sordidus is not well understood. The objective of the current work was to assess the effects of treating banana plants with F. oxysporum V5w2 and with nutrients on plant attractiveness and suitability for C. sordidus. We also investigated whether banana root inoculation with B. bassiana G41 would alter the attractiveness of plants to C. sordidus.
Materials and methods An overview of the experiments We investigated the effect of F. oxysporum V5w2 and B. bassiana G41 on the preference of C. sordidus adult weevils to banana plants through three laboratory experiments: F. oxysporum V5w2 with intact plants (experiment 1) or with corm pieces from plants grown with or without nutrients (experiment 2), and B. bassiana G41 with corm and pseudostem pieces (experiment 3). We also studied the effect of F. oxysporum V5w2 and plant
36 Effects of endophytic Fusarium oxysporum V5w2 as influenced by host….. nutrition on host suitability of intact banana plants for C. sordidus larvae in the screenhouse (experiment 4), and on host choice and oviposition preference of C. sordidus in the field (experiment 5).
Banana plants East African highland banana plants (cv. Kibuzi) for the experiments with F. oxysporum V5w2 were obtained through tissue culture from the laboratory of IITA Sendusu, Uganda. The plants had been produced by the shoot-tip micropropagation technique (Vuylsteke 1998), and maintained for six weeks on nutrient solution (1g/L, Polyfeed™, Haifa Chemicals, Israel). The endophyte F. oxysporum V5w2 (nit3) had been developed by Paparu et al. (2009a), and stored in sterile soil culture at 4 ºC at the laboratory of IITA Sendusu. The F. oxysporum V5w2 inoculum comprised of a suspension that contained ~1.5 × 106 spores/mL, prepared according to Paparu et al. (2006a). The banana plants were randomly assigned to two treatments (2 × 2), and therefore four groups, viz. banana plants with and without F. oxysporum V5w2, and treated with fertilizer (nutrient solution) or not (nutrient-deficient). Plants were inoculated by dipping the roots in the spore suspension of F. oxysporum V5w2 for 4 h (Paparu et al. 2006a). The plants were grown in steam-sterilized loamy soil that was contained in 2.5 L plastic pots. All plants were treated with 100 mL of the nutrient solution once a week until 8 weeks, at which they had attained a suitable girth size (~3 cm diameter) for weevil infestation. Water (100 mL per plant) was supplied daily. Nutrient deficiency was induced by not providing nutrient solution for four weeks. Nutrient deficiency was verified
37 Chapter 3 using a SPAD 502 chlorophyll meter (Spectrum Technologies Inc., USA), which indirectly measures leaf nitrogen content in SPAD units (Reeves et al. 1993). The previously mentioned plant treatments were made in four sets: two groups for the two laboratory host choice experiments (experiment 1 and experiment 2), a group for host suitability experiment (experiment 4), and a group for the host choice and oviposition preference field trial (experiment 5). Tissue culture plants of the East African highland banana (cv. Mpologoma) were used for the experiment on the effect of B. bassiana G41 on host preference in adult weevils (experiment 3), which were supplied either as untreated controls, or already inoculated with the entomopathogen according to Akello et al. (2007).
Banana weevils Adult C. sordidus were collected from old banana fields at IITA, Namulonge using pseudostem traps (Tinzaara et al. 2005). Weevils were sexed on the basis of punctation patterns on the rostrum and the angle of inclination of the 9th abdominal segment (Gold et al. 2004b). The insects were reared on banana corms and allowed to produce larvae for experiment 4.
Experimental dark room A dark room (5 3 m) was prepared for experiments 1, 2 and 3. The windows and the door of the room were sealed with black polythene sheets. Temperature in the room varied between 22-28°C. A red Philips 40W bulb
38 Effects of endophytic Fusarium oxysporum V5w2 as influenced by host….. illuminated all experiments, which facilitates observations without disturbing insect behaviour (Tinzaara 2005). An electric fan provided aeration during the experiments.
Olfactometers Olfactometer A (Figure 3.1) was designed for experiment 1 to investigate the response of banana weevils towards intact banana plants. This apparatus consisted of two plastic containers, with a removable walking arena of gauze, connected by a bridge. Plants were placed in the containers without
1222 cmcm 20 cm 10 cm
x d d 3.5 cm b c c
33.5 30cm cm a a 30 cm
20 10cm cm
Figure 3.1. A two-choice olfactometer A that was designed for intact plants in Experiment 1: Plastic containers for placing the potted plant (a); plastic bridge (b); removable walking arena comprised of a plastic rim (c) and gauze (mesh size 0.6
mm) at the bottom (d). An insect was placed at point X, equidistant from the two arenas. A wet filter paper was placed on the plastic bridge and replaced after every tested weevil.
39 Chapter 3 removing them from their pots, and their volatiles could enter the walking arena through the gauze. A tested insect was placed on the bridge equidistant from the two arenas. A wet filter paper was placed on the plastic bridge and replaced after every tested weevil. A two-cup pitfall olfactometer B (Figure 3.2) was designed for choice experiments with plant tissues for experiment 2. Nine identical units of this apparatus were constructed to make it possible for running experiments with several pairwise treatments simultaneously. The olfactometer consisted of a 20 L bucket filled with soil to a quarter of the height (10 cm). Two replaceable 100 mL plastic cups were eccentrically dug into the soil 15 cm apart, and were 2 cm from the wall of the bucket. Plant material was placed in the cups. A small depression for placement of insects was made at the opposite end of the bucket equidistant (20 cm) from the two cups.
a
Figure 3.2. A two-cup pitfall olfactometer B that 15 cm was designed for Experiment 2: Bucket of 20 L c 40 cm c (a), soil (b), 100 mL plastic cups (c), depression for placement of insects (X). Nine identical units 20 cm 20 cm of this apparatus were constructed to make it
x possible for running experiments with several 10 cm pairwise treatments simultaneously. b
30 cm
40 Effects of endophytic Fusarium oxysporum V5w2 as influenced by host…..
A two-choice olfactometer C was constructed based on those that were used by Lofgren et al. (1983) and Tinzaara et al. (2005) (Figure 3.3). This olfactometer was preferred for its stringency in comparing two types of plant material in experiment 3. The apparatus consisted of a Petri dish arena with a lid (9 cm in diameter) that sat on a tripod stand. Two holes were made at the sides of the dish to fit the arms of the olfactometer i.e. tubes of 5 mm diameter. The arms were each connected to an Erlenmeyer flask (500 ml) that contained the plant material i.e. corms or pseudostems (40-50 g). A wet filter paper was placed on the bottom of the Petri dish at the start of each test. Volatiles could enter the arena by diffusion from the plant parts in the Erlenmeyer flask.
Experiment 1: Assay with intact banana plants on the effect of F. oxysporum V5w2 on host preference in adult weevils Experimental comparisons that were assessed by using olfactometer A (Figure 3.1) included: (a) volatiles from control plants vs. clean air, to verify whether the weevils could respond to the untreated plants (n = 100 weevils), (b) volatiles from F. oxysporum V5w2-treated plants vs. clean air, to verify whether the weevils could respond to the endophyte-treated plants (n = 100 weevils), and (c) volatiles from control plants vs. volatiles from F. oxysporum V5w2-treated plants, to compare attractiveness between the two groups of plants (n = 50 weevils). After introducing a female weevil in the experimental setup, it was continuously observed for 15 min and then discarded. An insect was considered to have responded when it entered one of the walking arenas, or when it was within 1 cm from the entry point of
41 Chapter 3 the arena at the end of the observation period. Insects that did not show any movement within 2 min of the start of the experiment were considered to be non-responsive. The apparatus was washed with ethanol (70 %) and water between each trial run.
Experiment 2: Assay with pieces of banana plants on the effect of F. oxysporum V5w2 and nutrients on food preference in adult weevils Pieces of corms (≥ 10 g) were obtained from banana plants and allowed to self-ferment for 48 h in the dark room to enhance their attractiveness, before they were placed in the plastic cups. Weevil responses to the corms were assessed in different paired-treatment combinations in olfactometer B (Figure 3.2). The treatments consisted of corms obtained from plants that had received a PolyFeed™ nutrient solution (N+), no nutrients but only water (N-), or both previous treatments either inoculated with the endophyte (E+) or not inoculated (E-), and an empty cup (O). For example, N+/E+ were plants treated with both the nutrient solution and the endophyte. The treatments in the following nine combinations were used to assess weevil preference: (1) O vs O, (2) N-/E- vs N-/E-, (3) O vs N-/E+, (4) N+/E- vs N- /E-, (5) N+/E- vs N-/E+, (6) N-/E+ vs N+/E+, (7) N-/E- vs N+/E+, (8) N- /E+ vs N-/E-, and (9) N+/E- vs N+/E+. There were nine identical olfactometers, one for each combination. Female weevils were starved for 24 h to enhance their motivation to search for food. A weevil was placed at the depression in the bucket while corms from different treatments were placed inside the cups (Figure 3.2). The bucket was covered during each trial to provide darkness and prevent external odour interference. After 3 h,
42 Effects of endophytic Fusarium oxysporum V5w2 as influenced by host….. the presence of a weevil inside one of the two cups, or within 2 cm from the cup’s border, was recorded as the preferred choice. The trial was repeated 42 times i.e. six insects per treatment combination were assessed every day for 7 days. A fresh insect and piece of corm was used in each experimental run, while exchanging the positions of treatments in successive runs. The cups were cleaned with distilled water and ethanol after every trial.
Experiment 3: Assay with pieces of banana plants on the effect of B. bassiana G41 on food preference in adult weevils This experiment assessed the discrimination of C. sordidus between pieces from banana plants inoculated with B. bassiana G41 and controls using olfactometer C (Figure 3.3). Weevils were individually tested (n = 100) using either corms or pseudostems. The movement of every insect was monitored continuously for 15 min. An insect was considered to have respo-
9 cm
a d d c c
b
21 cm e e
34 cm
Figure 3.3. A two-choice olfactometer C that was designed for Experiment 3: Petri dish (a), tripod stand (b), holes (c), arms i.e. tubes of 5 mm diameter (d), Erlenmeyer flask (e). This olfactometer was preferred for its stringency in comparing two types of plant material. The apparatus was based on those described by Lofgren et al. (1983) and Tinzaara et al. (2005). 43 Chapter 3 nded when it entered one arm of the olfactometer, or when at the end of a 15 minute-exposure period the female was at < 1 cm from the entry point of the arms. Insects that did not show any movement within 2 min of the start of the experiment were discarded. After testing 6 individuals the arms of the olfactometer as well as the odour sources were replaced to compensate for any unforeseen asymmetry in the setup.
Experiment 4: Effect of F. oxysporum V5w2 and nutrients on host plant suitability for C. sordidus larvae The experiment comprised of four treatments (N-/E-, N-/E+, N+/E-, N+/E+) each having 15 plants replicated three times i.e. 45 plants per treatment, hence a total of 180 plants. Plants within each treatment replicate were grouped together to facilitate the application of the fertilizer treatment, and the 12 groups were randomized in the screenhouse. In the rearing, C. sordidus larvae were gently removed from corm pieces, weighed and placed separately in labelled glass Petri dishes containing wet filter paper to help minimize desiccation. Potted banana plants were infested by making a downward notch at the juncture of the pseudostem and the corm using a sterile surgical blade, then placing a larva into the notch (Figure 3.4). Larvae that did not start burrowing into the plants within 30 seconds were discarded and replaced with fresh ones. The notches were then covered with masking tape to ensure that the larvae remained inside the plant and to protect them from adverse conditions. All the 180 plants from the four treatments were completely randomized in the same screenhouse after infesting them with the weevils.
44 Effects of endophytic Fusarium oxysporum V5w2 as influenced by host…..
The insects were allowed to grow for 30 days during which a weekly supply of the nutrient solution was only given to those plants that were assigned the nutrient treatment, and water supplied daily to all plants. Plant heights and girths were recorded after 30 days. At harvest, the plants were gently removed from the pots and their roots were cut off. Peripheral damage (PD) was estimated by paring the corms, and then marking them vertically into four approximately equal sections that represented 25% (quarter) of the surface area. The amount of corm surface tissue consumed by weevil larvae in each quarter was estimated and the sum scores for the four sections expressed as a percentage. The corms were split transversely
3.43.1
Figure 3.4. Cosmopolites sordidus larva being infested into a notch at the base of a potted tissue culture banana plant (encircled). into two pieces. Internal injury on the lower corm was estimated on 25% sectors as percentage cross sectional area covered by damaged tissues on corm (CD). The number of tunnels on the lower corm was counted as an
45 Chapter 3 estimate of tunnelling damage (TD). During damage assessment, larvae that were inside the corms and in soil were carefully recovered. The larvae were weighed using an analytical balance (Denver Instrument SI-234, Denver, Colorado, USA), and the number of recovered insects expressed as a percentage of the initial sample size.
Experiment 5: Effect of F. oxysporum V5w2 and nutrients on host choice and oviposition preference of C. sordidus under field conditions The experiment comprised of four treatments (N-/E-, N-/E+, N+/E-, N+/E+) each having 10 plants replicated three times. Three strips (20 m long, 2 m wide) of land were prepared, spaced 30 m from each other. In each strip, 20 banana plants were put in holes, without removing them from their pots, in two rows, 1 m apart, and at 1 m distance in the row (Figure 3.5, 3.6). The plants were arranged in a way that an endophyte-treated plant and its corres-
N OE NE O
1 m ******
NE O N OE
1 m Figure 3.5. A portion showing the arrangement of potted banana plants (circles) in three strips of land in the field. This portion was repeated five times, resulting in a
continuous arrangement of 20 plants on both sides of the strip. The treatments included banana plants fertilized with nutrient solution only (N); with nutrient solution and endophytic F. oxysporum V5w2 (NE); unfertilized (O); and unfertilized but with the endophyte. The asterisks represent the nine points per strip on which adult C. sordidus were released.
46 Effects of endophytic Fusarium oxysporum V5w2 as influenced by host….. ponding control within a nutrient treatment were paired between the rows. This arrangement was repeated ten times while exchanging the positions of paired treatments subsequently within the strip. A thin layer of dry grass was laid at the base of each plant to create conducive environment for the insects. Thirty-two weevils (20 females and 12 males) were released in the centre of each quartet of plants late in the evening. The next morning (12 h after release), the number of weevils at the base of each plant was counted. Weevils were allowed to lay eggs for five days. After five days the plants were removed from the holes and the number of weevils attached to the pseudostems counted. Damage on the plants, caused by the weevils, was categorized as high or low (i.e. high >x; low < x; x = a quarter of pseudostem basal circumference damaged). Weevil eggs were counted by gently scraping corm and pseudostem tissues using a surgical blade. Weevil numbers counted from plants at each end of the strips were not included, due to lower chance of attracting the insects.
a b
Figure 3.6. (a) Arrangement of potted banana plants in a strip of land in the field, and (b) released adult C. sordidus comprised of aggregated 20 female and 12 male weevils (encircled; represented by the asterisks in Diagram 3.4).
47 Chapter 3
Statistical analysis Data were analyzed using SAS 9.1 software. Data for the two-choice assays in experiments 1, 2 and 3 were analyzed by χ2 test in proc freq to compare the outcomes against the expected 50% distribution. Friedman’s two-way analyses of variance for ranks and LSmeans in proc glm were used in comparisons for chlorophyll content and plant damage by the weevil (PD, TD, CD) between treatments in experiment 4; and for number of eggs in experiment 5. Two-way ANOVA with LSD was used for comparison of plant heights girth and larval weights between treatments. The percentages of recovered insects on plants in experiment 4 and experiment 5, and pseudostem damage in experiment 5 were analyzed using proc genmod as data having binomial distribution; Bonferroni-adjusted p-values were obtained by proc multtest from raw p-values in proc genmod and used for pairwise comparisons between treatments in experiment 4. Pearson correlations between larval weights and other parameters in experiment 4 were obtained using proc corr.
Results In experiment 1, which compared intact plants for the effect of F. oxysporum V5w2 on response of weevils in olfactometer A, a significantly higher number of weevils chose control plants than clean air (χ2 test, p < 0.05; Figure 3.7). The weevils in experiment 1 did not discriminate between clean air and F. oxysporum V5w2-treated plants (χ2 test, p = 0.46), and did not discriminate between controls and F. oxysporum V5w2-treated plants (χ2 test, p = 0.53) (Figure 3.7).
48 Effects of endophytic Fusarium oxysporum V5w2 as influenced by host…..
In experiment 2, which compared banana corm pieces for the effect of F. oxysporum V5w2 and nutrients on food choice by weevils in olfactometer B, a significantly higher number of insects preferred banana corms (without nutrients and with the endophyte) to clean air (χ2 test, p < 0.05), but the insects did not discriminate between corms of different treatments in all other comparisons (χ2 test, p > 0.05) (Figure 3.8).
Control plants 81 89 Fusarium-treated
Clean air 41 48 Fusarium-treated
Clean air * 26 55 Control plants
80 60 40 20 0 20 40 60 80
% weevils moving to odor source
Figure 3.7. (Experiment 1). Preference of the banana weevil C. sordidus between intact plants treated with endophytic F. oxysporum V5w2 and control plants. A total of 200 weevils were used in the experiment, 170 insects that were responsive were considered for analysis. Bars represent percentages (and figures numbers) of weevils choosing either of the odor sources (Asterisks* indicate significant difference, χ2 test, p < 0.05).
In experiment 3, which compared banana corm pieces for the effect of B. bassiana G41 on the response of weevils in olfactometer C, significantly lower numbers of insects chose sections from plants that were inoculated with the entomopathogen than those that were not inoculated (χ2 test, corms p = 0.04, pseudostems p = 0.03; Figure 3.9).
49 Chapter 3
Experiment 4 compared the effects of F. oxysporum V5w2 and nutrients on suitability of banana plants as hosts for larval survival and performance of C. sordidus in the screenhouse. As an indicator of host-plant quality, chlorophyll content was assessed. The chlorophyll level was higher in fertilizer-treated plants than in nutrient-deficient ones (Table 3.1). In fertilizer-treated plants, those that were inoculated with F. oxysporum V5w2 had a lower chlorophyll level than the ones without the endophyte. However
N+/E- 11 21 N+/E+
N-/E+ 12 20 N-/E-
N-/E- 15 22 N+/E+
N-/E+ 19 16 N+/E+ N+/E- 21 16 N-/E+ N+/E- 19 18 N-/E-
O 5 28 * N-/E+
N-/E- 14 21 N-/E-
O 12 14 O 70 60 50 40 30 20 10 0 10 20 30 40 50 60 70 80 90 100 % weevils making choice
Figure 3.8. (Experiment 2). Preference of the banana weevil C. sordidus between pieces of banana corms derived from plants treated (E+) and not treated (E-) with endophytic F. oxysporum V5w2, either fertilized with nutrient solution (N+) or unfertilized (N-), and empty cups (O). For example,
N+/E+ are plants treated with both the nutrient solution and the endophyte. Each pair of bars is an independent experiment conducted with a total of 42 weevils (only the responsive insects were considered). Bars represent percentages (and figures numbers) of weevils choosing either of the corms (Asterisks* indicate significant difference, χ2 test, p < 0.05).
50 Effects of endophytic Fusarium oxysporum V5w2 as influenced by host….. in nutrient-deficient plants, there was no difference in chlorophyll level between endophyte-treated and the untreated plants. Plants that were inoculated with F. oxysporum V5w2 suffered significantly less damages than those that were not inoculated (Table 3.1). Fusarium oxysporum V5w2-inoculated plants were taller and had wider girths than those that were not inoculated. Also, at the end of the experiment, fertilizer-treated plants were significantly taller and thicker than those without nutrients. The percentages of recovered larvae between the four treatments were not significantly different (fertilizer only 46.7% n = 45, fertilizer + endophyte 24.4% n = 45, nutrient-deficient 48.9% n = 45, nutrient-deficient + endophyte 42.2% n = 45) (χ2 test, df = 3, p = 0.07). Initial larval weights did
Control * 49 30 Beauveria-treated
Control * 49 31 Beauveria-treated
80 60 40 20 0 20 40 60 80
% weevils moving to odor source Corms Pseudostems Figure 3.9. (Experiment 3). Preference of the banana weevil C. sordidus between pieces of
corms and pseudostems from banana plants inoculated with the entomopathogen B. bassiana G41 and controls. Non-responsive weevils that were not considered for analysis were 21 for corms and 20 for pseudostems. Bars represent the overall percentages (and figures numbers) of weevils choosing either of the odor sources (Asterisks* indicate significant difference, χ2 test,
p < 0.05).
51 Chapter 3 not vary between the treatments (Table 3.1). However, at the end of the experiment, larvae from fertilizer-treated plants had significantly lower weights than those from nutrient-deficient plants. Larvae from F. oxysporum V5w2-treated plants tended to have lower weights than those from untreated plants, but this was statistically non-significant (p = 0.07). Larval weights were negatively correlated with chlorophyll content (R = -0.24, p = 0.04). However, there was no correlation between larval weight with other measured parameters (number of inner and outer tunnels, percentage damage of inner and outer corm, and percentage peripheral damage) (p > 0.05). In experiment 5, we compared intact banana plants for the effect of F. oxysporum V5w2 and nutrients on host choice and oviposition preference of C. sordidus in the field. At 12 h after insect release, a lower number of weevils was found on plants that had been inoculated with F. oxysporum V5w2 than on non-inoculated plants (p < 0.0001; Figure 3.10). The total number of weevils found on plants at the end of the experiment (i.e. after 5 days) was 263, their distribution did not vary between treatments (χ2 test, df = 3, p = 0.26). There was no difference in percentage of plants that had high pseudostem damage between the four treatments (fertilizer only 33.0% n = 30, fertilizer + endophyte 20.0% n = 30, nutrient-deficient 36.7% n = 30, nutrient-deficient + endophyte 23.3% n = 30) (χ2 test, df = 3, p = 0.42). There was no difference in number of eggs (mean = 3) that were found on plants between treatments (F test, p > 0.05).
52 Effects of endophytic Fusarium oxysporum V5w2 as influenced by host…..
Table 3.1. (Experiment 4) Chlorophyll content (chloro), corm damage by C. sordidus larvae, plant size and larval weight in tissue culture banana plants either inoculated with the endophyte F. oxysporum V5w2 or not, and either treated with fertilizer (nutrients) or not under screenhouse conditions
Source of variation df Chloroc Corm damage by larvae Plant size Larval weight Peripheral Internal Tunnels Height Girth Initial Final
F values
Endophyte (Fo) 1 1.5 16.4*** 17.8*** 9.8** 111*** 58.6*** 0.5 3.3 Nutrient (N1) 1 296*** 1.8 1.8 0.2 486*** 474*** 0.4 8.8** Fo*N 1 9.0* 0.1 0.4 0 0.1 0.8 0.4 0
Total (df) 3trt 176e 176e 176e 176e 175e 175e 176e 69e
Means
SPADs2 percent percent count cm cm gram gram Grand 51.7 27.3 12.9 2.5 34.9 9.1 0.01 0.12
Endophyte Fo 51.4 21.2b 8.8b 2.2b 37.9a 9.3a 0.01 0.11 non 52.0 33.4a 16.9a 2.9a 32.1b 8.8b 0.01 0.12
Nutrient fertilizer 57.8 29.2 10.3 2.5 40.9a 9.6a 0.01 0.10b non 45.7 25.4 15.5 2.5 20.0b 8.6b 0.01 0.13a
Endophyte fertilizer 56.5 b 25.3 7.2 2.1 43.6 9.8 0.01 0.09 non 46.3 c 17.1 10.5 2.2 32.3 8.9 0.01 0.12 non fertilizer 59.1 a 33.1 13.3 3 38.3 9.5 0.01 0.11 non 45.0 c 33.8 20.4 2.8 25.8 8.2 0.01 0.14
1 Fertilizer used was PolyFeed™ nutrient solution, Haifa chemicals, Israel; 2 SPAD units (Reeves et al., 1993); trt Treatment and e Error degrees of freedom; c Chlorophyll and corm damage (Friedman’s ANOVA & Lsmeans); plant height and larval weight (ANOVA & LSD). Means for significant effects are indicated in bold font; those with the same letter are not significantly different (p > 0.05), Asterisks indicate the level significance (***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05)
53 Chapter 3
35 Figure 3.10. (Experiment 5) ab a 134 Distribution of 458 Cosmopolites 30 131 sordidus weevils on banana plants bc 25 105 treated with fertilizer or water only, c with or without Fusarium oxysporum 88 20 V5w2, 12 hours after release of 1824 insects under field conditions. Bars 15 represent the overall percentages (and numbers above bars are the actual 10 populations) of weevils on the four
plant treatments; those with the same Distribution of weevils on plant treatments (%) 5 2 letter are not significantly different (χ 0 test, p > 0.05). fertilizer only fertilizer + water only water + endophyte endophyte
Discussion In the current study, the number of adult weevils was lower on plants that had been inoculated with F. oxysporum V5w2 compared to those without the endophyte 12 h after release of the insects in the field (experiment 5). However, in the olfactometer, the weevils did not discriminate between clean air and plants inoculated with F. oxysporum V5w2, but chose intact control plants against clean air (experiment 1). Future studies may investigate whether low preference of intact endophyte-infected banana plants by C. sordidus results from changes in volatile emission due to in planta infection with F. oxysporum V5w2, or whether odours that interfere with host perception by the weevil are emitted when the fungus colonizes the rhizosphere.
54 Effects of endophytic Fusarium oxysporum V5w2 as influenced by host…..
The behaviour of C. sordidus varies under different experimental conditions. We tested the effects of F. oxysporum V5w2 on the behaviour of weevils towards intact banana plants (experiment 1) and pieces of their corms (experiment 2) when compared to clean air. The insects did not discriminate between intact F. oxysporum V5w2-treated plants and clean air. However, the weevils chose cups containing corm sections from F. oxysporum V5w2-treated plants against empty cups. Results from the two experiments may not be fully comparable as the apparatus used were different. However, corm sections may have emitted more or stronger volatiles as they were injured and fermented plant parts, which could have resulted in a clear response by the weevils. Fermented banana plant sections have previously been shown to be more attractive to C. sordidus than fresh ones (Tinzaara et al. 2005, 2007). Furthermore, F. oxysporum V5w2 instigated short-term effects on the behaviour of adult C. sordidus. This was evident in experiment 5, where the number of weevils was low on F. oxysporum V5w2-treated plants 12 h after release of the insects. Yet at the end of the experiment there was no difference in the number of weevils, their eggs and pseudostem damage between treatments. In the present study, F. oxysporum V5w2 expressed the potential of suppressing plant damage by C. sordidus larvae. This was clear in experiment 4, whereby plants that were inoculated with F. oxysporum V5w2 exhibited lower corm damage by C. sordidus larvae than those that were not inoculated, and were larger in terms of height and girth. Our results concur with the suggestion by Niere et al. (2004) that some strains of F. oxysporum could limit the damage of banana plants by C. sordidus larvae. Larvae that
55 Chapter 3 grew in F. oxysporum V5w2-treated plants tended to have lower weights than those from untreated plants. Niere et al. (2004) also indicated that inoculation of banana plants with some strains of F. oxysporum could constrain the growth of C. sordidus larvae. The artificial endophyte B. bassiana G41 influenced the behaviour of C. sordidus towards banana plants. This is because fewer weevils were attracted to corm and pseudostem pieces from plants that were inoculated with B. bassiana G41 compared to those that were not inoculated with the fungus (experiment 3). Organic nitrogen content in plant tissues is important for insect growth (Mattson 1980; Schoonhoven et al. 2005). In the present study, nitrogen content was low in nutrient-deficient plants as indicated by the low chlorophyll level. However, larvae from the nutrient-deficient plants had greater weights than those from fertilizer-treated plants. This contradicted our expectation that nutrient-deficient plants are less suitable food for insect growth. However, there was no difference in larval feeding damage between fertilizer-treated and nutrient-deficient plants. Possible explanations for the low larval weights from fertilizer-treated plants include: (1) richness in nutrients with low concentrations of defensive compounds (Bryant et al. 1987), which allowed faster larval development to surpass the growth peak that is characterized by pre-pupal weight loss (Ochieng'-Odero 1990), earlier than those from nutrient-deficient plants, (2) fertilizer enhanced the synthesis of plant defensive compounds that limited larval growth (Herms 2002), and (3) fertilizer instigated imbalance in plant nutrient content
56 Effects of endophytic Fusarium oxysporum V5w2 as influenced by host….. leading to the accumulation of toxic inorganic compounds such as nitrates (Mattson 1980). In the present study, the behaviour of C. sordidus was not influenced by host plant nutrition. For example, the weevils did not discriminate between fertilizer-treated and nutrient-deficient intact banana plants under field conditions (experiment 5). Similarly, the weevils did not discriminate between corm pieces from fertilizer-treated and nutrient-deficient banana plants (experiment 2). Also, the number of eggs that the weevils laid did not vary between plants under the two nutrient regimes (experiment 5). It is likely that the cues from banana plants that are utilized by C. sordidus were not affected by fertilizer application. However, plant nutrition has been found to affect host preference in some curculionid insects. For example, Mao et al. (2001) found that the weevil Cylas formicarius elegantulus laid fewer eggs on sweet potato plants under high N fertilizer application than those with low N. The current studies were conducted using sterilized soil, the applicability of these results to natural conditions in which there are numerous soil microbes needs to be investigated. Also, changes in soil properties that may have been caused by sterilization and fertilizer application were not quantified, but would have been a basis for comparison of the current results with those of other studies. Furthermore the observations cannot be sufficiently attributed to in planta or rhizosphere activities of the two endophytes since we did not check for their presence in plant tissues and in the soil. Although endophyte-treated and endophyte-free plants were tested against clean air for attractiveness to the banana weevil to
57 Chapter 3 depict monoculture situations, the applicability of such olfactometer tests in the field was not assessed. Based on the results of the current work, we conclude that, under sterile soil conditions, inoculating tissue culture banana plants with F. oxysporum V5w2 and B. bassiana G41 may interfere with host infestation by C. sordidus, and may suppress corm damage by their larvae. Larval C. sordidus grow better on nutrient-deficient plants, but soil fertility did not affect host-derived cues for the adults.
Searching for weevils in plants and soil (Ssebaggala)
Acknowledgements This work was conducted under the management of the International Institute of Tropical Agriculture (IITA) in Uganda. Financial support was provided by the German Ministry of Economic Cooperation and Development (BMZ), and Wageningen University and Research Centre (WUR). We thank Dr. William Tinzaara of Bioversity International for his contributions in experimental designs. We acknowledge Patrick Emudong, Jane Luyiga, Phillip Abidrabo, Juliet Akello and Victoria Naluyange for the technical assistance. Thanks to the Ugandan National Banana Research Programme and Dr. Caroline Nankinga for supplying B. bassiana G41 used in this study.
58 Chapter 4
Comparative studies on effects of NPK nutrient deficiencies and soil sterility on banana plants and Radopholus similis infection
Team work during harvesting of plants
Dennis M.W. Ochieno · Marcel Dicke · Thomas Dubois · Danny Coyne · Piet van Asten · Arnold van Huis
Chapter 4
Abstract Root parasitism by nematodes such as Radopholus similis may vary under different plant nutritional conditions and soil substrates. Such variations in nematode infection may affect the deductions made from experiments that evaluate pest control measures. We conducted three factorial experiments in the screenhouse to investigate the effects of deficiencies in nitrogen (N), phosphorus (P) and potassium (K) and soil sterility on banana growth and R. similis infection. Plants were larger in non-sterile than in sterile soil; but were smaller when N-deficient solution or water only was applied. Compared to complete nutrient solution (CNS) treatment, R. similis density was higher and root death lower in plants that received N-deficient solution or water only, but not different when P-deficient and K-deficient solutions were applied. Compared to CNS, root necrosis was higher in P-deficient and lower in K-deficient plants, but not different in plants treated with N- deficient solution or water only. Root damage and R. similis density were lower in non-sterile than in sterile soil. Plants inoculated with R. similis were larger and appeared greener than those without the nematode when treated with N-deficient solution. We conclude that banana root infection by R. similis varies depending on plant nutritional status and soil sterility. Such variations need to be considered when evaluating technologies that target R. similis in potted banana plants.
60 Comparative studies on effects of NPK nutrient deficiencies…………….
Introduction Radopholus similis is a serious nematode pest of banana plants worldwide (Marin et al. 1998). This nematode damages banana roots by burrowing through the cortex while restlessly migrating within the tissues in search of new host cells. Such feeding and migratory habits of R. similis result in necrotic lesions and death of roots. Root damage caused by R. similis interferes with nutrient uptake while weakening the anchorage, which results in poor growth and toppling of banana plants (Gowen et al. 2005). Tissue culture is being employed for the multiplication of pest-free banana planting material (Vuylsteke 1998). Plants produced by tissue culture are grown in potted sterilized soil to maintain their pest-free condition before being transferred to farmers. In an effort to develop other pest management options, potted banana plants grown in sterilized soil have often been used for experiments on technologies that target R. similis e.g. plant resistance (Viaene et al. 2003) and microbial endophytes (Paparu et al. 2009b). However, results from studies that utilize sterilized soil may not address changes in soil chemical properties caused by sterilization, and the role of rhizosphere microbes that occur in the field (Troelstra et al. 2001). Also, nutrient depletion is usually high in pots and may affect results. In the case of R. similis, it may be difficult to identify the best condition under which a control measure works when populations of the nematode and its infection symptoms vary due to experimental conditions. The current study aimed at clarifying the effects of soil microorganisms and nutrients on banana plants and R. similis. The objective
61 Chapter 4 of this study was to evaluate the effects of soil sterility, nitrogen (N), phosphorus (P) and potassium (K) on plant growth and R. similis infection.
Materials and methods Experimental design
Table 4.1. Treatments in three screenhouse experiments that investigated the effects of soil microorganisms and nitrogen, phosphorus, potassium on banana plant growth and R. similis infection Experiment* Treatments Nematode Soil Nutrient solutions CNS1 N-deficient2 P-deficient3 K-deficient4 Water5 Exp. N yes Sterile x x x Non-sterile x x x no Sterile x x x Non-sterile x x x Exp. P yes Sterile x x Non-sterile x x no Sterile x x Non-sterile x x Exp. K yes Sterile x x Non-sterile x x no Sterile x x Non-sterile x x
1Complete nutrient solution (Murashige and Skoog 1962); either 2nitrogen, 3phosphorus or 4potassium was omitted from CNS, 5no nutrients were applied (See Table 4.2); ‘x’ represent the nutrient treatments that were considered in the three experiments respectively; *Each experiment had three replications
Three screenhouse experiments namely Exp. N (nitrogen), Exp. P (phosphorus) and Exp. K (potassium) were conducted with potted banana plants at the International Institute of Tropical Agriculture (IITA), Namulonge, Uganda. Exp. N, Exp. P and Exp. K investigated the effects of deficiencies of each of the three nutrients in banana plants respectively, and how they affect the infection by the nematode R. similis in sterile and non- sterile loamy soil. The three factorial experiments had plants that were inoculated with R. similis or left uninoculated, both having been grown in sterile or non-sterile soil. The experiments simulated plants that were either
62 Comparative studies on effects of NPK nutrient deficiencies……………. well nourished, receiving a solution with one nutrient deficient, or those receiving only water (i.e. no nutrients) (Table 4.1). Chemical compositions of the three nutrient solutions that were used in the experiments are shown in Table 4.2.
Banana plants and nematode inoculum Tissue culture banana plants (genomic group AAA-EA, cv. Kibuzi) were obtained from IITA, Namulonge, Uganda. The plants had been micropropagated by the shoot-tip culture technique in which plantlets are produced by multiplication of the meristematic tissue of banana corms (Vuylsteke 1998). The banana plants had been maintained for six weeks in nutrient solution (1 g/L, Polyfeed™, Haifa Chemicals, Israel). They were graded into four sizes based on their initial heights, and from each height category, plants were randomly distributed to groups that corresponded to the number of treatments within respective experiments, which varied between 14 to 16 plants between replicates. Initial fresh weights and number of healthy roots for every plant were recorded. Radopholus similis originated from banana fields at IITA Namulonge. The nematodes were maintained and multiplied on aseptic carrot discs (Speijer and De Waele 1997). Nematodes were supplied in sterile water suspension that contained mixed stages of 250 R. similis / mL.
Soil Loamy soil was collected from the soil surface (0-10 cm from) of a 5-year old unmulched banana field at Namulonge. The soil was sieved (5 mm
63 Chapter 4 aperture) before being thoroughly mixed. Soil samples were analyzed for chemical properties at the National Agricultural Research Organization (NARO), Kawanda, Uganda. Available P and exchangeable K were extracted using the Mehlich-3 method (Mehlich 1984). Phosphorus (P) in the extract was determined using the molybdenum blue colorimetric method and K using a flame photometer (Okalebo et al. 2002). Total N was analyzed by Kjeldahl oxidation and semi-micro Kjeldahl distillation (Bremner 1960). Organic matter (OM) was determined using the Walkley- Black method (Walkley 1947). Soil pH was analyzed using deionized water with a soil to water ratio of 1:2.5. The soil contained N (0.15 %), P (3.6 ppm), K (0.38 cmolc / kg), OM (2.4 %) and had a pH of 5.1. Part of the loamy soil was steam-sterilized at 100ºC for 1 h using an electrode steam conditioner (Marshall-Fowler, South Africa). Analysis of soil chemical properties was not done after sterilization.
Planting and inoculation with nematodes The banana plants were grown in 2.5 L buckets containing either sterile or non-sterile soil under screenhouse conditions (25 ± 3ºC, 70-75 % RH, 12L: 12D photoperiod). The containers had been perforated at their bases with 6 holes. Initial plant height, plus length and width of the youngest open leaf were recorded. Plants in two replicates of Exp. N, Exp. P and Exp. K were completely randomized in the screenhouse. Plants in all experiments were daily supplied with rain water (100 mL), and a weekly supply of 100 mL of the respective nutrient solutions (Table 4.2). Twenty days after planting, three holes (5 cm deep) were made into the soil around the base of each
64 Comparative studies on effects of NPK nutrient deficiencies……………. plant, by using a disinfected stick. Each plant was inoculated with ~500 R. similis in 2 mL of the nematode suspension distributed across the three holes before covering them with soil.
Table 4.2. Chemical composition of nutrient solutions in milligrams per litre of water Chemical Nutrient solutions CNS* N-deficient P-deficient K-deficient
NH4NO3 1650 . x 2402
KNO3 1900 . x . KCl . 401 93 .
CaCl2.2H2O 440 x x x
MgSO4.7H2O 370 x x x
KH2PO4 170 x . .
NaH2PO4.H2O . . . 171 KI 0.83 x x x** x x x H3BO3 6.2 x x x MnSO4.4H2O 22 x x x ZnSO4.7H2O 8.6 x x x Na2MoO4.2H2O 0.25 x x x CuSO4.5H2O 0.025 x x x CoCl2.6H2O 0.025 x x x Na2EDTA.2H2O 37.3 x x x FeSO4.7H2O 27.8 *Complete nutrient solution that was formulated by Murashige and Skoog (1962); the other three solutions were made by omission of N, P or K from the CNS respectively. ** In the K-deficient treatment this negligible amount of potassium (K) was retained due to unavailability of alternative source of iodine (I). ‘x’ means the chemical is present in same quantity as CNS
Data collection At 100 days after nematode inoculation, plant growth parameters, i.e. height, number of leaves, length and width of youngest open leaf were recorded as described by Paparu et al. (2009b). Plants were harvested and the numbers of dead and healthy roots were recorded and used to calculate the percentage dead roots. The shoots and roots were detached and their
65 Chapter 4 fresh weights recorded. Root and shoot biomasses were combined to obtain total fresh weight. The shoots were oven-dried at 70ºC for 14 days to determine dry weight. The roots were preserved at 4ºC within three days of harvesting for assessment of necrosis and estimation of R. similis density based on the methods described by Speijer and DeWaele (1997) and Brooks (2004). General observations on the appearances of plants under different treatments were noted.
Statistical analysis Data were analyzed using SAS 9.1 software (SAS Institute Inc, 2001). Diagnostic check for normality was conducted using proc univariate. Proc transreg was used to find appropriate Box-Cox transformations, which included generation of suitable powers or lambda (λ) for data (Y) that required transformation. In Exp. N, lambda (λ) for root biomass was estimated as 0.75 hence the transformation became Y0.75; lambda was 1.5 for leaf length and width, 0.25 and 1.25 for shoot dry weight and plant height respectively; no transformation was done on shoot fresh weight, total biomass and number of leaves. In Exp. P, lambda (λ) was 0.75 for shoot fresh weight, 1.75 for number of leaves, 2.25 and 2.5 for leaf length and width respectively; and 1.5 for shoot dry weight, total biomass and plant height; no transformation was done on root biomass. In Exp. K, lambda was 0.5 for root biomass, shoot dry weight and total biomass; 0.75 for plant height, 2.25 for number of leaves, 2 and 1.5 for leaf length and width respectively; no transformation was done on shoot fresh weight.
66 Comparative studies on effects of NPK nutrient deficiencies…………….
In all experiments, percentage dead roots and necrosis (x) were arcsine√(x/100) transformed, while untransformed data were used for R. similis density. Proc glm was used for three-way factorial analyses of variance (ANOVA) among the treatments. Mean separation in proc glm was done by t-tests (LSD) when there were significant differences among treatment means.
Results General observations In Exp. N, plants treated with the N-deficient solution were stunted with yellow leaves that had pinkish veins; leaves of the plants dried from the margins towards the midribs, and wilted from the lower older ones, towards the upper young ones (Figure 4.1a). In Exp. N, nematode-treated plants appeared greener and taller than those without nematodes among the groups that received N-deficient solution (Figure 4.1b). In Exp. N, plants subjected to total nutrient starvation by receiving water only were stunted with some yellowing and drying leaves (Figure 4.1c). In the three experiments, plants treated with CNS remained green and appeared to be healthy when grown in non-sterile soil. However, leaves of most CNS-treated plants grown in sterile soil developed yellowish margins (Figure 4.2a, b). In Exp. N, soils that received N-deficient solution or water only became heavy and sticky by the time of harvest. In Exp. P, plants that received P-deficient solution had dark-green leaves with slightly yellowing laminae and margins, while the older leaves curled downwards and dried with dark coloration (Figure 4.3). In Exp. K, plants that had been fertilized with K-deficient solution
67 Chapter 4 developed bright yellow-orange colorations on leaf lamina (Figure not provided). Initial plant growth-related parameters did not vary significantly between the treatments in the three experiments (F test, P > 0.05), and have been summarized in Table 4.3.
Table 4.3. Initial growth-related parameters (mean ± SE) for tissue culture banana plants that were used in the three experiments.
Growth-related parameters Experiment N Experiment P Experiment K Weight 8.6 ± 0.2 7.1 ± 0.2 8.0 ± 0.2 Height 5.4 ± 0.1 5.5 ± 0.1 5.5 ± 0.1 Number of leaves 3.3 ± 0.0* 3.7 ± 0.0* 3.3 ± 0.0* Leaf length 12.5 ± 0.2 11.4 ± 0.2 12.8 ± 0.3 Leaf width 4.8 ± 0.1 4.2 ± 0.1 4.9 ± 0.1 Number of functional roots 3.6 ± 0.1 3.7 ± 0.3 3.7 ± 0.3 *SE≤0.05
Experiment N (Table 4.4) Nematode-treated plants had significantly higher percentage dead roots, necrosis and R. similis density than those not treated with nematodes. Nematode-treated plants grown in sterile soil had significantly higher percentage dead roots than those from non-sterile soil, when treated with CNS or water only; but this was not significant when treated with N- deficient solution. Percentage dead roots was higher in nematode-treated plants from sterile soil that received CNS, compared to those treated with N- deficient solution and those supplied with water only, the last two not being different. There was no difference in percentage dead roots between the three nutrient treatments among nematode-treated plants that were grown in non-sterile soil. Percentage root necrosis was higher in the nematode-treated plants that were grown in sterile than those grown in non-sterile soil, when
68 Comparative studies on effects of NPK nutrient deficiencies…………….
4.1a 4.1b 4.1c
4.2a 4.2b 4.3
Figure 4.1(a). Symptoms of nitrogen starvation in potted tissue culture banana plants that had been grown in steam-sterilized loamy soil and treated with N-deficient solution; the leaves turned
yellow and dried from margins towards the midrib, and wilted from the lower older ones towards the upper younger ones. Figure 4.1(b). Comparison between untreated plants (4 left) and those that were treated with the nematode Radopholus similis (4 right) when fertilized with N-deficient solution in steam-sterilized loamy soil; the nematode-treated plants appear to be taller and greener than those without nematodes. Figure 4.1(c). Symptoms of nutrient starvation in tissue culture banana plants that had been grown in steam-sterilized loamy soil and subjected to total
nutrient starvation by giving them water only; the leaves were yellowish and dried in a similar manner as in the N-starved ones but to a lesser extent. Figure 4.2 (a, b). Yellowish coloration on leaf margins of potted tissue culture banana plants that were grown in steam-sterilized loamy soil and fertilized with a complete nutrient solution (CNS). Figure 4.3. Symptoms of phosphorus starvation in potted tissue culture banana plants that had been grown in steam-sterilized loamy soil and treated with P-deficient solution; the leaves were green but developed yellow coloration on laminae and margins, they curled downwards and dried with a dark colour. supplied N-deficient solution or water only; but not when fertilized with CNS. Percentage root necrosis was not different between the three nutrient treatments among the nematode-treated plants in sterile and in non-sterile soil. Roots from sterile soil had significantly higher R. similis density than those from non-sterile soil in plants that were treated with N-deficient
69 Chapter 4 solution or water only; but not different when fertilized with CNS. In sterile soil, R. similis density was significantly lower in roots that were fertilized with CNS, than those that were treated with N-deficient solution and water only, the last two being similar. Density of R. similis in roots from non- sterile soil was not different between the three nutrient treatments. Root biomass was significantly lower in plants that were treated with R. similis compared to those without the nematode in sterile and in non- sterile soil. Among the nematode-free plants, root biomass was significantly higher in those from sterile than from non-sterile soil. Among the nematode- treated plants, root biomass was not different between plants from sterile soil and those from non-sterile soil. In non-sterile soil, root biomass was significantly higher in plants that were fertilized with CNS than those that were treated with N-deficient solution, which had more root biomass than those that were given water only. Root biomass in sterile soil was not different between the three nutrient treatments. Plants in sterile soil had significantly more root biomass than those from non-sterile soil when treated with water only; but were not different when fertilized with CNS or N-deficient solution. In sterile and non-sterile soil, plants that were treated with CNS had a higher shoot fresh weight than those supplied with N-deficient solution, and lowest in plants that were given water only. Within each of the three nutrient treatments, plants in sterile soil had significantly more shoot fresh weight than those from non-sterile soil. Within each of the three nutrient treatments, shoot dry weight was not different between plants that were inoculated with R. similis and those without the nematode. Among the
70 Comparative studies on effects of NPK nutrient deficiencies……………. nematode-treated plants, those that were fertilized with CNS had a higher shoot dry weight than those that received N-deficient solution, and was lowest in plants that were given water only. Among the nematode-free plants, shoot dry weight was higher in plants that were fertilized with CNS than those that received N-deficient solution or water only, the last two not being different. In sterile and in non-sterile soil, shoot dry weight was significantly higher in plants that received CNS compared to those treated with N-deficient nutrient solution or water only, the latter two not being different. Shoot dry weight was significantly higher in plants that were grown in sterile than in non-sterile soil when under the supply of water only; but was not different between the soils when CNS or N-deficient solutions were applied. Plants that were treated with R. similis had fewer leaves than those without the nematode when grown in sterile soil. However, the number of leaves did not vary between nematode-treated and nematode-free plants in non-sterile soil. The number of leaves was lower in sterile than in non- sterile soil when R. similis was present. However, in the absence of the nematode, the number of leaves was not different between sterile and non- sterile soil. Both in sterile and in non-sterile soil, plants that were fertilized with CNS produced significantly more leaves than those treated with N- deficient solution or water, the latter two not being different. Among plants that were fertilized with CNS, the number of leaves was significantly higher in non-sterile than in sterile soil; such differences were not evident under the supply of N-deficient solution or water only.
71 Chapter 4
Total plant biomass was significantly lower in plants that were treated with R. similis than in those without the nematode. Leaf widths were not different between nematode-treated and nematode-free plants in sterile and in non-sterile soil. In nematode-free plants, leaves were narrower in sterile than in non-sterile soil; but the widths were not different between the soils in nematode-treated plants. Nematode-treated plants that received N- deficient solution were taller and had larger leaves than those without nematodes. However, among plants that received CNS or water only, plant height and leaf size were not different between plants treated with and without nematodes. In sterile and non-sterile soil, plants that were treated with CNS had the highest total biomass and leaf size, then those supplied with N-deficient solution, and lowest in plants that were given water only. Plant height had similar trends as total biomass between nutrient treatments in non-sterile soil; but slightly differed by having equivalent values between N-deficient solution and water only in sterile soil. Among plants that received N-deficient solution or water only, those from sterile soil were taller, had higher total biomass and larger leaves than the ones from non- sterile soil. Total plant biomass, height and leaf size were not different between sterile and non-sterile soil when CNS was applied.
Experiment P (Table 4.5) Dead roots, root necrosis and R. similis were mainly found in plants treated with the nematode. Nematode-treated plants that were grown in sterile soil had significantly higher percentage dead roots and R. similis density than those from non-sterile soil. Nematode-treated plants that were fertilized with
72 Comparative studies on effects of NPK nutrient deficiencies…………….
P-deficient solution had significantly higher percentage root necrosis than those treated with CNS. Root biomass was significantly lower in nematode-treated plants from sterile soil than in those from non-sterile soil; but in nematode-free plants, root biomass was not different between the two soils. In sterile soil, root biomass in nematode-treated plants was significantly lower than in plants without nematodes. However, in non-sterile soil, root biomass was not different between plants that were treated with R. similis and those without the nematode. Plants that were inoculated with R. similis had lower total biomass than those that were not inoculated with the nematode. Plants that were fertilized with P-deficient solution had lower shoot fresh weight and total biomass than those that were treated with CNS. None of the treatments or their interactions affected shoot dry weight. In sterile soil, plants that were inoculated with R. similis had significantly fewer leaves than those without the nematode; however in non- sterile soil, there was no difference. In both nematode-treated and nematode- free plants, more leaves were recorded in non-sterile than in sterile soil. In sterile soil, plants that were fertilized with P-deficient solution had significantly fewer leaves than those treated with CNS. However, in non- sterile soil, there was no difference in number of leaves between plants that were fertilized with P-deficient solution and those that were given CNS. Within both CNS and P-deficient nutrient treatments, plants that were grown in non-sterile soil had more leaves than those from sterile soil.
73 Chapter 4
3***
Height Leaves Leaves Height 29.2 28.6 4.2 4.3 34.6 34.0 15.9 15.6 Length Number Width fwt
194.7 b 212.4 a Total dwt
values Shoot fwt
151.7 153.9 17.4 17.3 Shoot fwt Plant biomass
Root grams grams grams grams cm count cm cm cm count grams grams grams grams -100g Count a 15678 5188b a 54.8 46.0 b a 171.7 133.6b a 18.8 15.8 a b 226.5 a 30.1 180.0 b b 4.1 27.7 b a 35.5 4.4 a a 16.1 33.0 b 15.5 b R. similis F
35.5 ab 5104b 56.5a 227.1a 26.6 a 283.6 a 39.4 a 5.1 a 44.2 a 20.5 a percentage 35.9 36.3 33.8 b 14773a 51.6a 131.5b 13.3 b 183.9 b 24.8 b 3.8 b 31.2 b 14.3 b Necrosis
39.3 a 11441a 43.1 b 98.6 c 11.9 c 141.7c c22.2 3.8 b 27.0c 12.4c ,soil sterility, and type ofnutrient solution (nitrogen-deficient, CNS, water) on root damage, nematode density, 20.8 a a 20.8 13.8 b Death Means a 32.5 b 2.1 a 62.7 b 9.0 a 19568 b 1010 b 43.0 a 57.9 17.4 36.1 10422 50.4 152.8 17.3 203.5 28.9 4.2 34.3 15.8
11 486 481 475 486 487 484 486 482 482 481 482 1 87.7*** 61.8*** 33.9*** 2 10.1** 6.7** 0.6 9.1*** 0.1 2.8 3.2 3.1 0.3 0.3 10.2** 3.7 2.5 8.0** 1.1 1.0 0.7 1.0 0.9
49.1 44.5 189.9 33.5 26.5 10.3 28.4 22.8 18.1 19.0 20.7 2 2 4.2* 2.4 3.6* 0.6 10.2*** 0 4.7** 14.4*** 1.9 4.1* 3.2* 11.0*** 0.6 7.3*** 14.2*** 16.7*** 4.7* 10.4*** 18.4*** 1.2 5.5** 9.1***
15.7 3 19.7 Radopholussimilis 2 R. similis Water 16.6 ) 1 1333.7*** 1335*** 106.5*** 57.5*** 0.2 1.1 10.3** 1.6 0.6 3.2 3.0 1
Rs Effectsof × S × N × N ×S Soil Sterile Non-sterile Nutrient CNS N-def N-def Source of variation variation of Source df damage Root Main effects Nematode ( c.v. Total (df) percentage Nematode no Grand mean Soil sterility (S) Nutrient(N) type interactions Two-way 1 Rs 2 Rs 12.4*** N × 3.1 S 0 interactions Three-way 3.6* Rs 33.2*** 19.9*** 9.9*** 123.7*** 17.5*** 460.6*** 57.1*** 89.3*** 205.7*** 28.5*** 269.1*** 10.2** 511.1*** 49.5*** 149.9*** 12. 708.7*** 622.8***
plant biomass, height, number and size of leaves in potted tissue culture banana plants plants banana culture 4.4. Table tissue potted in of leaves size and number height, biomass, plant
74 Comparative studies on effects of NPK nutrient deficiencies……………. 16.0 ab 15.9 ab 16.2 a 15.0 b Treatment trt trt 27.6 d cd12.8 30.1 c 30.1 c13.5 43.8 a 20.3 a 26.4 d 12.0 d 32.4 b 15.0 b 44.5 a 20.6 a 35.5 33.6 35.6 32.3 0.05); ≤ 0.01, *p ≤ 3.9 3.8 5.1 3.7 3.8 5.1 4.0b 4.4a 4.2a 4.3a 0.001, **p 22.7 c22.7 23.4 c23.4 39.4 a 21.7 c21.7 26.2 b 39.4 a ≤ 153.3 188.5 293.1 130.5 179.2 274.1 12.6 bc 12.3 bc a 26.7 11.2 c c 11.2 b 14.3 a 26.6 169.8 133.4 173.6 19.5 133.7 15.2 213.9 18.1 175.2 16.4 30.3 239.1 28.1 184.9 29.9 27.4 50.0 103.4 59.5 127.5 64.1 229.1 36.5 94.0 43.6 135.7 49.0 225.1 30060 a 30060 9160 b bc 44.1 c 937 41.9 c c 1083 a 65.5 50.1b b 6599 20834a 53.9ab 19288a 58.9240.2 aba b 3715 51.7ab 156.3 c 26.7a b 8786 59.0a 120.4 d b 2982 15.3b 44.2b 294.1 a 214.3 b 14.6b 33.7c 215.2 b 38.9 a 106.7 d 26.6a 172.1c 26.9 b 4.7 b 74.9e 11.2bc 24.6 cb 3.9 273.344.3 a a 8.9 152.2c c c3.8 33.0 20.0 b a 39.9 a c22.7 c 29.6 108.615.0 b d 5.5a c3.8 c13.4 19.4 d 44.0 a c 29.5 c3.8 20.9 a c13.6 24.1 d 11.3 d 12856 12856 40432 39.9 36414 47.7 5209 235.2 44.7 16541 158.8 57.9 5560 116.4 28.9 39.4 182 215.1 16.3 27.8 1713 275.1 112.5 13.4 68.3 877 206.6 70.6 24.3 69.8 2556 39.2 161.1 12.2 245.3 58.7 644 28.0 273.0 9.0 4.6 153.9 60.1 196 23.7 151.9 124.4 3.7 24.5 49.0 39.6 44.0 98.4 3.7 213.6 14.2 40.0 24.4 34.0 313.5 5.6 101.0 15.9 19.7 19.5 28.8 223.7 4.0 79.6 28.7 15.5 38.6 45.1 3.7 183.1 10.3 12.8 30.9 25.8 273.6 8.8 4.8 21.4 23.8 25.5 152.4 14.6 4.0 40.2 44.7 119.6 11.1 4.0 21.1 32.0 5.3 20.3 19.3 30.3 3.6 14.5 40.0 3.8 13.9 28.0 20.4 24.4 12.5 11.5 dryweight; Means forsignificant interactions areindicated bold in font; thosewith thesameletter arenot significantly dwt Nitrogen-deficient solution(N-starved); Asterisks indicate significant effect (***p 68.3 57.0 3.8 14.6 3 c 2.6 bc 12.6 c 550 c 1.3 c 4.7 c 1185 c 2.3 b 10.1 bc 1269 b 30.0 a 63.7 a 21544 30.3 ab ab 30.3 a 63.2 a 28195 a 41.0 b 24.0 0.6 d c 3.5 a 37.2 a 61.2 b 8939 a 50.4 b 35.6 abc 45.8 b 36.8 ab 53.2 c 24.0 a 56.3 bc 25.0 bc 41.8 c 22.9 c 38.5 e 0.3 c 36.1 de 1.3 d 2.6 e d 0.3 0.1 de 4.2 d 1.0 de d 1.2 4.4 d 3.4 d 5.3 d 12.8 Freshweight and fwt Complete nutrientsolution ; 2 ; ; N-def Water 18.2 N-def 18.6 Water 34.7 37.9 13.1 14.4 32.9 40.9 Sterile CNS Sterile CNS Error degrees of freedom; freedom; of degrees Error e Rs Radopholus similis
Water Water N-def N-def No nematode CNS Water Water N-def N-def and different (p > 0.05) 0.05) > (p different Nematode Sterile Non-sterile No nematode Non-sterile Sterile Nematode CNS Sterile CNS Non-sterile CNS 25.7 14.0 35.2 Rs 35.8 1 N-def Water Non-sterileCNS N-def no Water N-def Water Non-sterileCNS N-def Water
75 Chapter 4
Plants that were grown in non-sterile soil were taller and produced larger leaves than those from sterile soil. Leaves were significantly longer and wider in plants that were fertilized with CNS than those that were treated with P-deficient solution.
Experiment K (Table 4.6) Dead roots, root necrosis and R. similis were mainly found in plants that were treated with the nematode. Nematode-treated plants in sterile soil had significantly higher percentage dead roots and R. similis density than those from non-sterile soil. Plants fertilized with CNS had significantly higher percentage root necrosis than those treated with K-deficient solution. Root biomass was significantly lower in plants that were treated with R. similis than those without the nematode, within both nutrient treatments (CNS and P-deficient), and within both soils (sterile and non-sterile). Root biomass was greater in non-sterile than in sterile soil, in nematode-treated and in nematode-free plants, as well as within the two nutrient treatments. In nematode-treated plants, root biomass was significantly lower in those that were fertilized with K-deficient solution than those treated with CNS. However, in nematode-free plants, root biomass was not different between plants that were fertilized with CNS and those that received K-deficient solution. In non-sterile soil, root biomass was significantly higher in plants that were fertilized with CNS than those that were treated with K-deficient solution. However, in sterile soil, root biomass was not different between plants that were fertilized with CNS and those that were treated with K- deficient solution.
76 Comparative studies on effects of NPK nutrient deficiencies……………. ** 38.5 39.9 16.6 17.2
a 5.5 a 40.4 a 17.5 b 5.2 b 38.0 b 16.3
b 5.1 a 5.5
Height Leaves 35.4 33.4
33.8 35.1 32.6 b 32.6 b 4.8 b 37.7 b 16.0 36.3 a a 36.3 a 5.8 a 40.7 a 17.8 Number Length Width fwt 237.1a 216.2b 213.8b 238.7a Total dwt 18.1 17.0
values Shoot F fwt 183.3 a 183.3 165.4 b b 165.4 Means Means 167.8 180.4 17.3 17.8 172.2 17.7 219.1 176.3 17.4 233.9 Shoot fwt Root Plant biomass
grams gramsgrams grams cm count cm cm -100g
count R.similis 4261 53.8 3820 50.7
5021 a5021 46.9 b 3034 b3034 57.6 a b 14.1 a 20.1
16.9 17.5 , soil sterility, and type of nutrient solution (phosphorus-deficient, CNS) on root damage, nematode nematode damage, root on CNS) (phosphorus-deficient, solution nutrient of type and sterility, , soil 15.4 17.2 4036 52.2 174.2 17.6 226.5 34.4 5.3 39.2 16.9
Death Necrosis percentage percentage 31.0a 0.4b 34.6a 0.3b a7931 b210 46.0 b 58.3 a 19.4 a 19.4 11.6 b 16.8 14.0 3
2 7 338 334 333 338 338 338 338 339 339 339 338
1 1 1 48.5*** 3.5 0 1.6 1 12.2*** 12.1*** 7.5** 0.1 0.9 1.4 0.5 3.2 0 2.3 0.5 1.6 1.0 1.4 0 0.1 3.1 3.1 2.4 0.7 0.1 6.2* 0.5 0.2 1.6 2.6 0.7 0 2.7 0 0.3 1.3 6.2* 0.5 1.8 1.0 2.3 0.9 1.7 1.7 49.1 54.1 155.3 34.8 43.7 26.3 43.1 38.2 26.4 35.7 33.9
Radopholus similis R. similis ) 1 1335.3*** 1049.9*** 129.6*** 38.9*** 3.6 0.7 9.3** 1.5 16.0*** 2.1 3.2 1
Effects of of Effects Rs × S× N× N× × S P-def
Grandmean Soilsterility (S) Nutrient(N)type Two-way interactions 1 1 Rs Rs N× S 25.2*** 3.2 2.2 interactions Three-way 16.1*** Rs 8.8** 0.8 30.4*** 0.2 2.2 0.2 8.0** 2.8 3.2 13.7*** 6.6* 117.6*** 3.5 17.1*** 49.0* 11.1** 8.8** 18.1*** Source of variation variation of Source df damage Root Nematode ( c.v. Total(df)
Maineffects Nematode no Soil Sterile Non-sterile
Nutrient CNS density, plant biomass, height, number and size of leaves in potted tissue culture banana plants plants banana culture tissue 4.5. potted Table in leaves of size and number height, biomass, plant density,
77 Chapter 4 Treatment Treatment trt 0.05); 39.8 17.4 41.4 18.1 36.3 41.5 15.2 18.2 37.0 39.9 15.7 38.3 17.6 16.3 39.1 16.8 ≤ 0.01, *p ≤ 5.8 a 5.9 a 4.6 c 5.9 a 4.5 c 5.8 a 5.2 b 5.1 b 0.001,**p ≤ 179.4 17.2 240.4 37.6 162.7 173.0 17.0 181.4 17.7 200.6 18.4 227.1 32.6 236.9 34.9 32.5 383c 61.0 a 10119a 5716b 37.8 b 40c 54.1 a 55.6 a 8584 49.3 180.2 18.0 229.5 35.1 5.4 39.8 17.1 7330 190 42.9 58.0 156.4 186.3 16.7 18.3 199.2 244.3 32.5 32.5 4.9 5.7 37.2 40.9 16.2 17.9 230 58.6 174.5 17.3 233.1 34.3 5.4 38.8 16.4 dryweight; Means for significant interactionsboldfont;with those same letter the indicated are not in are significantly 0.5 33.8 35.4 0.1 b 28.7 a 40.1 c 0.3 c 0.4 dwt Phosphorus-deficient solution (P-starved); Asterisks indicate significant effect (***p (***p effect significant indicate Asterisks (P-starved); solution Phosphorus-deficient 3 0.8 c c 0.8 39.7 a 39.7 b 22.6 c 0 Fresh weight and and weight Fresh
fwt 33.2
28.7
P-def 12.1 20.3 2755 57.1 172.4 17.0 229.5 36.0 P-def 21.5 19.9 4862 44.4 158.5 17.1 202.9 30.8 P-def 0.4 CNS 17.2 13.6 5188 49.4 186.4 18.3 235.8 34.4 Complete nutrient solution; 2 ; Errordegrees of freedom; e Radopholus similis Non-sterile CNS 11.0 14.5 3323 58.2 180.3 17.9 238.4 36.5 different (p > 0.05) and Nematode Sterile Non-sterile No nematode Non-sterile Sterile Nematode CNS Sterile 1 P-def No nematode No CNS 0.3
78 Comparative studies on effects of NPK nutrient deficiencies…………….
In both sterile and non-sterile soil, nematode-treated plants had significantly lower shoot fresh weight and total biomass than those without nematodes. In nematode-treated plants, those that were grown in non-sterile soil had significantly higher shoot fresh weight and total biomass than those from sterile soil. However, in nematode-free plants, shoot fresh weight and total biomass were not different between plants from the two soils. In plants that were fertilized with CNS, those from non-sterile soil had significantly higher shoot fresh weight and total biomass than the ones from sterile soil. Conversely, in plants that received K-deficient solution, shoot fresh weight and total biomass did not vary between the soils. In non-sterile soil, plants that were treated with CNS had significantly higher shoot fresh weight and total biomass than those that were treated with K-deficient solution. However, in sterile soil, shoot fresh weight and total biomass were not different between the nutrient treatments. Nematode-treated plants that were grown in sterile soil and fertilized with CNS had lower shoot dry weight than those without nematodes. Such differences in shoot dry weight were not evident in non-sterile soil, and among plants that were fertilized with K-deficient solution. Shoot dry weight did not vary between sterile and non-sterile soil. In both nematode-treated plants and nematode-free ones, those from non-sterile soil were significantly taller and had more leaves than the ones from sterile soil. In sterile soil, nematode-treated plants were shorter and had fewer leaves than those without nematodes. However, in non-sterile soil, plant height and number of leaves were not different between nematode-treated plants and those without nematodes.
79 Chapter 4
19.3 19.8 42.3 b 43.6 a
Height Leaves 38.1 5.9 39.5 5.9 Length Number Width fwt
231.0 b 270.4 a 36.3 b 41.3 a 5.5 b 6.3 a 40.8 b 45.1 a 18.2 b 20.8 a Total dwt
20.5 19.6 Shoot F values F
fwt Shoot fwt 51.2 b 186.8 b 19.1 b 238.0 b Plant biomass Root 56.5 a 206.5 a 20.9 a 263.0 a grams grams grams grams cm count cm cm -100g 5132 R. similis 3756
6117a 2798b 46.5 b 61.1 a 184.5 b 209.3 a 11.6 b Necrosis 15.6 a
14.1 13.3 , soil sterility, typeand of nutrient solution (potassium-deficient, CNS) on root damage,nematode density, 9.2 9.2 13.7 4422 54.0 197.1 20.1 251.0 38.8 5.9 43.0 19.5 Death percentage percentage count 18.9 a 0.1 b 27.9 a 0.4 b 8710a 358b 47.3 b 60.3 a 179.7 b 213.4 a 18.4 b 21.7 a 226.9 b 273.6 a 36.4 b 41.1 a 5.7 b 6.1 a 41.3 b 44.6 a 18.9 b 20.1 a 12.1 a 6.5 b 1 1 13.0*** 0.7 1 20.0*** 1.0 5.2* 6.3* 3.8 2.9 0.1 6.0* 4.6* 0.1 5.4* 3.5 2.4 5.6* 1.7 1.9 1.0 1.5 1.2 0.5 1 1 0.0 1.0 3.3 2.8 2.5 0 148 5.3* 10.0** 0.1 Means 6.5* 97.8 1.6 0 179.2 0.3 7.3** 1.0 1.7 17.8 0.1 11.3*** 26.7 1.0 9.7** 16.3 1.1 6.4* 13.7 15.3 27.8 24.6 17.6
9.1 9.3 3 2 Radopholus similis Radopholus R. similis ) 1 178.8*** 341.5*** 103.8*** 51.9*** 39.3*** 21.2*** 43.4*** 30.7*** 9.7** 37.4*** 21.9*** 1 Rs ofEffects × S × N × N ×S
K-def Grand mean Source of variation variation of Source df damage Root Main effects ( Nematode Soil sterility (S) Nutrient(N) type Two-way 1 interactions 1 Rs Rs 13.1*** 0.0 N × S 0.1 interactions Three-way 4.2* Rs 14.4*** (df) 56.6*** 2.7 17.2*** c.v. 1.0 Total368 11 4.6* 365 364 12.4*** 29.7*** 368 366 363 7.2** 35.7*** 366 369 369 369 369 93.4*** 50.7*** 10.5** 117.7*** 2.3 1.0 Nematode 4.0* 3.3
no Soil Sterile Non-sterile Nutrient CNS Table 4.6. plant biomass, height, number and size of leaves in potted tissue culture banana plants banana culture tissue potted in of leaves size and number height, biomass, plant
80 Comparative studies on effects of NPK nutrient deficiencies…………….
Treatment trt 0.05); ≤ 38.8 17.4 43.9 20.4 42.8 18.9 46.3 21.2 41.1c 18.9c 41.5 bc 18.9 bc 46.1 a 20.6 a 43.1 b 19.6 b
0.01, *p ≤
0.001, **p 0.001, ≤ 36.4 36.3 5.3 42.3 5.7 41.2 6.4 40.4 18.3 45.9 18.1 21.2 40.1 6.2 44.3 20.5
199.1c 33.2 c 5.2 c 255.6b 39.8 ab 6.2 a 262.9ab 39.5 b 5.8 b 283.5a 42.6 a 6.3 a 233.1 b 228.9 b 289.7 a 247.6 b 198.1 200.1 32.1 280.9 34.2 223.2 5.0 40.5 268.1 5.4 38.9 257.6 37.9 6.3 40.6 298.0 39.7 6.2 38.3 17.1 267.4 44.1 5.5 44.0 17.7 43.6 6.0 41.1 20.5 44.4 6.4 20.3 41.2 6.2 19.4 47.5 18.4 44.9 21.7 20.6
18.1 18.6 22.9 20.5 21.6 19.5 20.3 18.8 17.8 b 18.5 b 19.6 b 17.3 b 25.4 a 20.5 b 21.0 ab 20.0 b
188.9 18.7 241.2 36.5 5.7 169.2 17.9 210.7 36.4 5.7 223.5 23.0 284.1 42.4 6.0 202.7 20.2 262.6 39.7 6.1
52.3 b 41.8 c 60.6 a 59.9 a 46.0c 47.0c 187.1 b 65.9 a 181.8 b 223.8 a 55.5 b 192.1 b
12168 a 38.6 c 160.5 c 5214 b 56.1 b 199.4 b 67c 54.4 b 208.4ab 623 c 65.6 a 217.9a
dry weight; Meansdry for significant interactions boldfont;with those are indicatedsame letter thein are not significantly 28.1 27.8 0.2 0.6
dwt Potassium-deficient solution (K-starved);Asterisks significant indicate (***peffect 3
24.0 a 13.7 b 0.1 c0.1 c0.1 Fresh weight and fwt
K-def 22.4 K-def 15.1 22.4 K-def 0 25.9 K-def 13701 0 0.2 36.5 6450 0.5 163.6 47.7 20 175.8 57.6 719 200.0 62.1 205.3 Complete nutrientsolution ; 2 ; ; Non-sterile K-def 19.0 24.0 10329 K-def 0 0.4 377 K-def K-def 11.2 7.0 11.3 11.9 6861 3324
Sterile CNS 25.7 33.8 10600 40.6 157.4 Sterile CNS 0.1 0.2 115 51.3 216.8 Errordegrees of freedom; e Rs Radopholussimilis different(p > 0.05) and Nematode Sterile 1 Non-sterile No nematode Sterile
Nematode CNS 18.9 31.4 7245
No nematodeNo CNS 0.1 0.5 340
Sterile Non-sterile CNS CNS 12.9 6.2 16.8 14.5 5357 2344 Rs No Non-sterile CNS 12.6 Non-sterile CNS 29.2 0.1 4225 0.6 63.0 217.9 535 68.7 229.3
81 Chapter 4
Leaf size (length and width) was significantly lower in nematode-treated plants than those without nematodes when fertilized with CNS. However, leaf size was not different between nematode-treated plants and those without nematodes when fertilized with K-deficient solution. In the absence of R. similis, plants that were treated with CNS had larger leaves than those that received K-deficient solution. However, in the presence of R. similis, leaf size was not different between plants that were fertilized with CNS and those that were treated with K-deficient solution. Leaves of plants that were grown in non-sterile soil were larger than those from sterile soil.
Discussion In the current study, plants that were treated with CNS exhibited better growth than those grown under the four nutrient deficiencies. N-deficient plants and those treated with only water appeared very stunted with yellowish leaves, which indicate a dominant N-deficiency symptom (Nachegowda et al. 1992; Uchida 2000). P-deficient plants had dark-green leaves with yellowish margins that curled downwards and dried. These are similar to some P-deficiency symptoms in banana described by Chatterjee and Dube (2004). K-deficient plants had bright yellow-orange colorations on the leaf laminae. This is a typical symptom of K-deficiency in banana plants (Sathiamoorthy and Jeyabaskaran 2001). Trends in Exp. P and Exp. K indicated better plant growth in non- sterile than in sterile soil for most parameters. Smith and Smith (1981) attributed improved growth of the legume Trifolium subterraneum in non- sterile soil to infection with vesicular-arbuscular mycorrhizal fungi.
82 Comparative studies on effects of NPK nutrient deficiencies…………….
However in Exp. N, plants grew better in sterile than non-sterile soil when N-deficient solution or only water were applied, which could be due to the elimination of soil microbial competition for N (Hodge et al. 2000), and enhanced availability of nutrients (Troelstra et al. 2001). In CNS-treated plants, those grown in sterile soil developed yellowish coloration on leaf margins, which may have been symptoms of nutrient deficiencies caused by soil sterilization, for instance iron deficiency (Evan et al. 2003). In the three experiments, R. similis multiplied and caused damage to banana roots. This resulted in lower root biomass and poorer growth compared to plants without the nematode. These are typical effects of damage to banana roots by R. similis (Viaene et al. 2003). Damage by R. similis was lower in non-sterile than in sterile soil in the three experiments; this may have been caused by nematode-suppressive activities of rhizosphere microbes in non-sterile soil (Kerry 2000). On the contrary, under the supply of the N-deficient solution, nematode-treated plants were taller and produced larger leaves with a greener appearance than those without nematodes. This may be an indication of improved N-acquisition by plants when R. similis is present in the soil. Plant-feeding nematodes may promote plant growth by improving soil N content through excretion of ammonia, defaecation and when their bodies decompose (Verschoor 2002). Root necrosis caused by R. similis was higher in P-deficient plants than those treated with CNS in Exp. P. This could be due to easy penetration of banana root tissues by the nematode, which is facilitated by secretion of cell wall degrading enzymes (Giebel 1982; Devi et al. 2009), through weak cell membranes, whose phospholipid composition (Helliot et al. 2003) may
83 Chapter 4 get altered under P-deficiency (Andersson et al. 2003). By contrast, the absence of K was associated with lower root necrosis compared to CNS treatment in Exp. K. These necrotic lesions were not related to R. similis treatment. Treatment of plants with N-deficient solution or water only did not cause differences in root necrosis when compared with CNS. The density of R. similis in roots was higher in N-deficient plants and those that received water only, when compared to CNS-treated ones.
This can be linked to the presence of nitrogen in the form of NH4NO3 in the CNS, which may be responsible for resistance against the nematode. Low + ammonium-nitrogen (NH4 -N) content in nutrient solutions has been associated with an increase in R. similis density in banana roots (Declerck et al. 1998). Banana plants utilize ammonium-nitrogen more efficiently than other forms of nitrogen (Nasir et al. 2003). This could be related to its easier assimilation into amino acids (Lam et al. 1996), which react with phenolic compounds to form pest-inhibitive quinine-amino conjugates under the influence of polyphenol oxidase (PPO) (Bittner 2006). Phenolic compounds and PPO are among factors that have been linked with resistance to R. similis in banana plants (Fogain and Gowen 1996; Valette et al. 1998; Wuyts et al. 2006). A higher percentage of dead roots was recorded in CNS-treated plants than those treated with N-deficient solution or water only. The question is why the resistance we have linked with the lower R. similis density in CNS-treated plants did not suppress root death. It is possible that plant treatment with CNS facilitated rapid death of root tissues infected with R. similis, through hypersensitive-like resistance reactions that occur in
84 Comparative studies on effects of NPK nutrient deficiencies……………. banana plants against the nematode (Mateille 1994). Omission of P or K seemed not to affect R. similis multiplication and root death, as the two parameters were not different between CNS-treated plants and those deficient of P or K respectively. Possibly, the presence of nitrogen in the form of NH4NO3 in the CNS or P and K-deficient solutions was responsible for the similar levels of R. similis density and root death. However, the contribution of other minerals to the observations on nematode densities and root death may not be excluded. Differences in chemical compositions between nutrient solutions are associated with variable effects that may not be well addressed in experiments (Clark 1982). We conclude that banana root infection by R. similis varied depending on plant nutritional status and soil sterility. Such variations need to be considered when evaluating technologies that target R. similis in potted banana plants.
Acknowledgements This work was conducted under the management of the International Institute of Tropical Agriculture (IITA) in Uganda. The funds were provided by the German Ministry of Economic Cooperation and Development (BMZ) and Wageningen University and Research Centre. Technical support that was provided by Jane Luyiga, Phillip Abidrabo, Patrick Emudong, Elvis Mbiru, Fred Kato, Victoria Naluyange, Rose Khainza and Juliana Nakintu. Dr. Phillip Ragama advised on statistical analysis. We give credit to the Soil Chemistry Laboratory of Kawanda Agricultural Research Institute (KARI) for the training and analysis of soil samples.
85
Chapter 5
Effects of soil sterilization on interactions between endophytic Fusarium oxysporum V5w2 and the root burrowing nematode Radopholus similis in tissue culture banana plants
Transporting harvested plants to the laboratory (Nteza and Victoria)
Dennis M.W. Ochieno · Marcel Dicke · Thomas Dubois · Piet J.A. van Asten · Danny Coyne · Arnold van Huis
Chapter 5
Abstract The potential of non-pathogenic Fusarium oxysporum V5w2 as an endophyte for biological control of the nematode Radopholus similis was investigated in sterile and non-sterile soil in pots, to assess the role of soil biota on results. Banana plants that were treated with R. similis and grown in sterile soil had a higher percentage of dead roots, a higher density of nematodes in roots, were shorter, had lower biomass, and produced fewer and smaller leaves, compared to those from non-sterile soil. Densities of R. similis in the rhizosphere were lower in non-sterile soil than in sterile soil. For all the investigated parameters, no differences were recorded between plants that were treated with R. similis only, and those co-inoculated with the nematode and F. oxysporum V5w2. Inoculation of plants with the endophyte, with R. similis, or with a combination of endophyte and R. similis resulted in a lower optical density of root extracts, which was similar for the three treatments. In vitro growth inhibition assays indicated the presence of Fusarium spp. and other fungi in the tissue culture banana roots; some of these interfered with the growth of F. oxysporum V5w2, while the endophyte negatively affected the growth of others. Our data show that soil sterilization eliminates plant growth promoting microbes and the natural suppressive effect of soil on R. similis in banana plants.
88 Effects of soil sterilization on interactions between endophytic Fusarium.…
Introduction The production of highland cooking banana (Musa spp.), an important staple food crop in Eastern Africa, is threatened by a complex of plant parasitic nematodes, especially Radopholus similis (Cobb) Thorne. This nematode species burrows into the root cortex of banana plants causing necrotic lesions and death of roots (Gowen et al. 2005). The consequences of root damage include impaired nutrient uptake and toppling of the plants. The annual average output of banana in a country like Uganda is 10.6 metric tons per hectare (Kikulwe et al. 2008). This is much less than the production potential, as banana is under the attack of a range of pests and diseases. In field trials, nematodes, including R. similis, were found to cause banana yield losses of up to 51% (Speijer and Kajumba 2000). In the face of the global rise in food demand, technologies that limit nematode damage in bananas and other food crops become increasingly important. Banana nematodes are readily transmitted through infested planting material. Therefore, using clean healthy material is an important strategy for minimizing nematode infestation and dissemination (Speijer et al. 1999). Besides paring and hot water treatment of suckers (Speijer et al. 1999), tissue culture is efficient for mass production of clean planting material. However, stringency in aseptic production of tissue culture banana plants not only eliminates harmful organisms, but also beneficial endophytic microbes (Sikora et al. 2000). Deficiency of protective endophytes renders tissue culture banana plants susceptible to reinfestation by nematodes in the field (Sikora et al. 2000). The risk of nematode reinfestation of clean
89 Chapter 5 planting material necessitates sustainable preventative methods such as using biological control agents. The International Institute of Tropical Agriculture (IITA) among other organizations investigated strains of non-pathogenic Fusarium oxysporum for reintroduction into tissue culture banana plants, to provide nematode control (Niere 2001; Athman et al. 2007; Paparu et al. 2008). Fusarium oxysporum V5w2 is an endophyte strain that has been considered to have biological control potential against R. similis (Vu et al. 2006; Athman et al. 2007). The actual mechanisms and conditions involved in the suppression of nematodes by non-pathogenic strains of F. oxysporum are not well understood and documented (Sikora et al. 2008). The mode of action appears to be mainly based on systemic induced resistance (Vu et al. 2006; Paparu et al. 2008; Sikora et al. 2008). As part of the process of producing tissue culture plants, the use of sterile soil is required, and at this stage endophytes are inoculated to provide them with optimal conditions for colonization. Most screenhouse evaluations of endophytic F. oxysporum on banana nematodes have been conducted using sterilized soil. However, soil is rich in microbes that interact with roots, endophytes and nematodes through e.g. symbiosis, parasitism, competition and antibiosis. Experiments that have been conducted in sterile soil may fall short of crucial information on the impact of soil microbes on the control of nematodes by endophytes. Soil sterilization can change nutrient availability in the soil (De Deyn et al. 2004). Also, some pot trials investigating the control of nematodes by F. oxysporum may have been conducted without replenishment of nutrients. As
90 Effects of soil sterilization on interactions between endophytic Fusarium.… depletion of nutrients in pots can be high, results consequently may not accurately reflect circumstances experienced in the field. Possible variations in the biocontrol potential of F. oxysporum V5w2 between studies may be better understood if the soil ecological factors under different experimental conditions and methodological approaches are assessed. The objective of the current study was to assess the impact of soil sterilization, and hence elimination of soil biota, on the control of R. similis by non-pathogenic F. oxysporum V5w2 under the supply of a complete nutrient solution. We hypothesize that in the presence of adequate nutrients, F. oxysporum V5w2, banana plants and soil biota interact positively to suppress detrimental effects of R. similis.
Materials and methods Experimental design The treatments, viz. nematode vs. no nematode, endophyte vs. no endophyte, sterile vs. non-sterile soil were studied in a full factorial experiment (2 × 2 × 2). This resulted in 8 treatments i.e. control, nematode, endophyte, and nematode+endophyte, each having been conducted in sterile and non-sterile soil. The treatments, each having 15 plants, were repeated nine times. The plants were completely randomized in the greenhouse in six out of nine replicates. One hundred days after the start of the experiment, data were collected on plant parameters that included (i) biomass (root, shoot dry weight, total biomass), (ii) percentage dead roots, (iii) percentage root necrosis, (iv) R. similis densities in roots and soil, (v) plant height, (vi) number of standing leaves, (vii) leaf length and width, (viii) identities of
91 Chapter 5 root-invading microbes and their interactions with F. oxysporum V5w2, and (ix) optical density of root extracts.
Banana plants Tissue culture banana plants (genomic group AAA-EA, cv. Kibuzi) were obtained from IITA, Namulonge, Uganda. The plants had been micropropagated by the shoot-tip culture technique in which plants are produced by multiplication of the meristematic tissue of banana corms (Vuylsteke 1998). The banana plants had been maintained for six weeks in nutrient solution (1 g/L, Polyfeed™, Haifa Chemicals, Israel), after which they were graded into four sizes and distributed to eight groups of 15 plants. Four groups were marked for inoculation with F. oxysporum V5w2 and the other four were unmarked. Initial fresh weights and number of functional roots of the plants were recorded.
Endophyte and nematode inoculum We used F. oxysporum strain V5w2 stored at IITA, which was originally isolated from banana roots and corms in Uganda (Schuster et al. 1995) and modified into a nit3 (KClO3-resitant) fungal strain by Paparu et al. (2009a). Inoculum was prepared by sprinkling the soil culture onto 90 mm diameter Petri dishes containing half-strength PDA (19 g/L, Sigma-Aldrich, Germany). Ten days later, the fungal spores and mycelia were scraped from the media into a 500 mL beaker containing sterile water. The spore mixture was sieved and homogenized, and then adjusted to 1.5 × 106 spores/mL.
92 Effects of soil sterilization on interactions between endophytic Fusarium.…
Radopholus similis were originally obtained from banana fields at Namulonge, IITA and maintained aseptically on carrot discs (Speijer and De Waele 1997). Nematodes were supplied in sterile water suspension containing mixed stages of 250 R. similis / mL.
Soil Loamy soil was collected within a depth of 10 cm from a 5-year old unmulched banana field at Namulonge. The soil was sieved (5 mm aperture) before being thoroughly mixed. Soil samples were analyzed for chemical properties at the National Agricultural Research Organization (NARO), Kawanda, Uganda. Available P and exchangeable K were extracted using the Mehlich-3 method (Mehlich 1984). Phosphorus (P) in the extract was determined using the molybdenum blue colorimetric method and K using a flame photometer (Okalebo et al. 2002). Total N was analyzed by Kjeldahl oxidation and semi-micro Kjeldahl distillation (Bremner 1960). Organic matter (OM) was determined using the Walkley-Black method (Walkley 1947). Soil pH was analyzed using deionized water with a soil to water ratio of 1:2.5. The soil contained N (0.15 %), P (3.6 ppm), K (149 ppm i.e. 0.38 cmolc / kg), OM (2.4 %) and 5.1 pH. Part of the loamy soil was steam- sterilized at 100ºC for 1 h using an electrode steam conditioner (Marshall- Fowler, South Africa). Analysis of soil chemical properties was not done after sterilization.
93 Chapter 5
Fungal inoculation, planting, and nematode infestation Plants that were marked for endophyte treatment were inoculated by the root-dipping technique (Paparu et al. 2006a), except that in the present study the roots were left undamaged. The roots were immersed into a 1.5 L spore suspension contained in tubs (30 × 25 × 15 cm, length × width × height) for 4 h. Control plants were immersed in distilled water. The banana plants were grown in 2.5 L buckets containing either sterile or non-sterile soil under screenhouse conditions (25 ± 3ºC, 70-75% RH, 12L: 12D photoperiod). Initial plant height, plus length and width of the youngest open leaf were recorded. The plants were supplied with rain water (100 mL) daily and a complete nutrient solution (CNS, 100 mL) weekly. One litre of CNS contained 1650 mg NH4NO3, 1900 mg KNO3, 440 mg CaCl2.2H2O, 370 mg MgSO4.7H2O, 170 mg KH2PO4, 37.3 mg
Na2EDTA.2H2O, 27.8 mg FeSO4.7H2O, 6.2 mg H3BO3, 22.3 mg
MnSO4.4H2O, 8.6 mg ZnSO4.7H2O, 0.83 mg KI, 0.25 mg Na2MoO4.2H2O,
0.025 mg CuSO4.5H2O and 0.025 mg CoCl2.6H2O (Murashige and Skoog 1962). Twenty days after planting, three holes (5 cm deep) were made into the soil around the base of each plant, by using a disinfected stick. Each plant was inoculated with 500 R. similis in 2 mL of the nematode suspension distributed across the three holes before covering them with soil. Initial plant growth parameters did not vary between the treatments, and they included fresh weight (7.9 ± 0.13 g), plant height (5.5 ± 0.08 cm), number of leaves (3.4 ± 0.02), leaf length (12.2 ± 0.10 cm), leaf width (4.6 ± 0.05 cm) and number of functional roots (3.6 ± 0.04) (n = 1084, mean ± SE, F test, p > 0.05).
94 Effects of soil sterilization on interactions between endophytic Fusarium.…
Data collection At 100 days after nematode inoculation, plant growth parameters (height, number of leaves, length and width of youngest open leaf) were recorded. Plants were harvested and the numbers of dead and healthy roots were recorded and used to calculate the percentage dead roots. The shoots and roots were detached and their fresh weights recorded. Root and shoot biomasses were combined to obtain total fresh weight. The shoots were oven-dried at 70ºC for 14 days to determine dry weight. The roots were preserved at 4ºC within three days. Five roots per plant were randomly selected for assessment of necrosis caused by nematodes, and for estimation of nematode densities. Three roots were randomly selected per plant to assess the presence of microbes. The remaining roots were used for colorimetric assessment (optical density) of their extracts. Root necrosis assessment and quantification of nematode densities were undertaken based on the methods of Speijer and De Waele (1997) and Brooks (2004). To investigate whether densities of R. similis varied in the rhizosphere between sterile and non-sterile soil, eight 100 mL soil samples were selected from each soil treatment. This was done for plants that were treated with R. similis only. Nematodes in the soil samples were extracted using a Baermann tray over a period of 48 h. The extract was concentrated to 25 mL followed by concurrent identification and absolute counts of nematode species in the sample. Each of the three roots selected for analysis of microbe infestation was surface-sterilized by dipping it into 96% ethanol followed by flaming. This ensured that only microbes within the tissues remained alive. Each root
95 Chapter 5 was then cut into six pieces. The root pieces were plated on 60 mm glass
Petri dishes containing PDA enriched with KClO3 for 7 days (Figure 5.1). Root-invading microbes growing on the root pieces were sub-cultured and identified at the Soil Microbiology Laboratory (Makerere University, Uganda). In vitro growth inhibition tests between the microbes and F. oxysporum V5w2 were conducted. The endophyte was inoculated in the centre of a Petri dish containing PDA using a sterile needle. The microbes were inoculated on four spots surrounding the endophyte (Figure 5.1). The endophyte or the test microbe was considered inhibitive if its mycelia grew into the colonies of the other marked with a boundary between them (Figure 5.1). For colorimetric assessments, five roots from three plants per treatment were chopped together into a composite sample. This was conducted in triplicate. Ten grams of each composite sample were mixed with 100 mL distilled water, and macerated using a Waring laboratory blender (Christison Scientific, Gateshead, UK). The root extracts were diluted (10×) and their absorbance measured at 320 nm using a spectrophotometer (Genesys 10 UV, Thermo Fisher Scientific, USA). Optical density of plant extracts has been used for assessment of cotton seedling infection by pathogenic F. oxysporum (Dong and Cohen 2002).
Statistical analysis Data were analyzed using SAS 9.1 software (SAS Institute Inc, 2001). Diagnostic check for normality was conducted using proc univariate. Since pre-analysis of data from the two sets of replicates yielded similar results,
96 Effects of soil sterilization on interactions between endophytic Fusarium.… they were all treated as completely randomized. Proc transreg was used to find appropriate Box-Cox transformations, which included generation of suitable powers or lambda (λ) for data (Y) that required transformation. For root biomass lamda (λ) was estimated as 0.75 hence the transformation became Y0.75. Lambda for plant height, leaf length and width was 2; and λ = 0.25 and 0.5 for data on shoot dry weight and optical density of root extracts respectively. Percentage dead roots and percentage necrosis (x) were arcsine√(x/100) transformed. No transformation was done on data for other parameters. Proc glm was used for three-way factorial analyses of variance (ANOVA) among the treatments. Mean separation was done by t-tests (LSD) when there were significant differences among treatment means. Mean separation of optical density of root extracts was done by Lsmeans with Bonferroni adjustment for multiple comparisons.
Results There was a significant difference in percentage dead roots among the treatments (p < 0.0001) (Table 5.1). Dead roots were mainly found in nematode-treated plants. The interaction between nematode treatment and soil sterility on percentage dead roots was significant (p < 0.0001): in nematode-treated plants, the percentage dead roots in sterile soil was significantly higher than those from non-sterile soil (p < 0.0001). Endophyte inoculation did not affect percentage dead roots (p > 0.05). There was a significant difference in the percentage root necrosis among the treatments (p < 0.0001) (Table 5.1). Percentage root necrosis was significantly higher in nematode-treated plants than in plants without
97 Chapter 5 nematodes (p < 0.0001). Endophyte treatment and soil sterility did not affect percentage root necrosis (p > 0.05). There was a significant difference in R. similis densities in roots among the treatments (p < 0.0001) (Table 5.1). Radopholus similis were mainly found in nematode-treated plants. Roots of nematode-treated plants from sterile soil had a higher density of R. similis than those from non- sterile soil (p < 0.0001). Endophyte treatment did not affect density of R. similis in roots (p > 0.05). Rhizosphere populations of R. similis (mean ± SE) in pots of nematode-inoculated plants were significantly higher in sterile soil (1346 ± 266, n=8) than in non-sterile soil (34 ± 8, n=8) (df = 1, F = 24.24, p = 0.0002). Thirteen other nematode species, identified mainly in non-sterile soil among the soil samples, included: the plant parasitic Helicotylenchus multicintus, Meloidogyne spp., Pratylenchus goodeyi, Xiphinema sp., Trophorus sp., Scutellonema sp., Rotylenchus sp., Tylenchus sp., Hemicycliophora sp., Paratrichodorus sp. and Ditylenchus sp. Saprophytic genera included Aphelenchus sp. and Rhabditis sp. among numerous others that we could not identify. There was a significant difference in plant biomass (root, shoot, total) among the treatments (p < 0.0001) (Table 5.1). Significant interactions on plant biomass existed between nematode and soil treatments (p < 0.0001): nematode-treated plants from non-sterile soil had more biomass than those from sterile soil. Shoot dry weight varied significantly between the treatments (p = 0.003) (Table 5.1); the main effects of nematode treatment (p = 0.001) and soil sterility (p = 0.003) on shoot dry weight were significant. Nematode-treated plants had low shoot dry weights. Plants from
98 Effects of soil sterilization on interactions between endophytic Fusarium.…
e
1047 e
1047 e
Leaves 5.4 5.4 41.6 18.9 5.9 ab 43.4b 20.0 a 5.6 5.6 43.9 19.8 5.2 b 43.2b 19.1 b 5.0 41.5 18.4 a 5.9 a 44.6 a 20.4 5.9 5.9 44.0 20.2 4.8c 39.8c 17.8c
1047
e V5w2 soil and sterility root on 36.5 b 36.5 38.2 39.4 a 39.4 38.2 36.5 b 36.5 40.5 39.4 a 39.4 34.7 dry weight dry font; those withthe same letter arenot significantly Number Length Width dwt fwt 1049 e 240.8 b 257.2 277.3 a 280.0 252.2 a 274.6 266.0 c 223.9 Total 1050 dwt e Fresh weight and and weight Fresh 22.3 a 22.3 20.9 b 20.9 Fusarium oxysporum fwt
Shoot fwt 1045 e values values 194.0 b 20.7 b 200.6 20.0 216.7 a 216.7 a 22.5 221.4 23.2 212.0 21.8 187.1 21.4 Shoot 1048 e fw t Biomass Height Root
Error degrees of freedom; of freedom; degrees Error e
gram gram gram gram cm count cm cm , the fungal, endophyte 1048 -100g e count R. similis R. similis 8770 47.7 5816 b 56.6 b 385 60.6 79 cd 58.6 ab 678 c 62.5 a 11853 a 11853 c 38.6 Treatment and and Treatment 1035 e trt Necrosis Necrosis percent 33.5 a 33.5 32.3 1.1 b 1.1 0.7 1.5 34.9 V5w 2; Radopholus similis 1044 e 0.6
14.3 17.2 4541 54.2 205.4 21.6 259.2 38.0 5.5 42.8 19.3
Death Means 28.4 19.0b
0.1 c 14.2 14.4 16.7 17.7 4763 4323 53.0 55.4 206.0 1.1 c 204.9 21.5 21.7 259.0 259.3 37.8 38.1 5.4 5.5 42.7 42.8 19.4 19.3 a 38.1 1 109.3*** 1 6.9** 2.1 35.7*** 31.5*** 1 0.4 10.3** 0.2 0.4 0.3 0.4 17.7*** 0.2 0 1.2 0 12.2**0.8 1.08.2** 5.6** 0.7 0.1 0.1 0.1 0.5 4.0 0 0.6 0 1.2 0.3 0.1 1 1.0 0.3 2.5 2.2 2.9 0 2.4 0.7 0.3 1.8 0.2 1 63.0*** 0.1 24.2*** 74.0*** 0.4 8.9** 9.2** 39.7*** 259.7*** 49.7*** 114.5***
1048 trt 74.3 73.7 197.5 30.1 28.1 9.7 28.8 33.3 23.6 24.1 26.6 7 F
Fusarium oxysporum oxysporum Fusarium end density, plant biomass, height, number and size of leaves in tissue culture banana plants treated with a complete plantswithtreatedin ofbanana height,leaves number complete a culture andplanttissue biomass,size density,
1 0.1 2.0 0.8 3.6 0.1 0 0 0.4 1.1 0 1.1 1 1694.0*** 1434.0*** 232.0*** 109.0*** 41.7*** 11.5** 64.2*** 40.7*** 14.9*** 44.5*** 25.8*** ) ) R . s im il is F. oxysporum oxysporum F. non Non-sterile Non-sterile 9.9 16.8 3223 59.6 206.4
Sterile 18.9 17.7 5919 48.7 204.5 n o end i nem Fo ut
Effectsof the nematode Rs l R. similis so × S
t
n Fo Fo e × Radopholus similis; S × × × S ri Fo Three-way interactions Rs c.v. (df) Total Nematode ( Rs Source of variation variation of Source df damage Root Maineffects ( Endophyte percent Nematode Non-sterile nem Soil (S) interactions Two-way Rs Grand mean mean Grand
Asterisks indicate significant effect (***p ? 0.001, **p ? 0.01, *p ? 0.05); Means for significant effects are indicated in bold in indicated are effects significant ?for *p ? Means **p 0.01, ? 0.05); 0.001, (***p effect significant indicate Asterisks different > 0.05) (p non nematode No Sterile Endophyte Soil
Nematode Sterile ut
damage, n Table 5.1.
99 Chapter 5 non-sterile soil had higher shoot dry weights than plants from sterile soil. The endophyte treatment did not affect plant biomass including the shoot dry weight (p > 0.05). There were significant differences in plant height, number of leaves, leaf length and width among the treatments (F test, p < 0.0001) (Table 5.1). The interactions of nematode and soil sterility on number of leaves, leaf length and width were significant (p < 0.05). Leaves of nematode-treated plants from sterile soil were fewer, shorter and narrower than for plants from non-sterile soil. For plant height, the main effects of nematode treatment and soil sterility were significant (p < 0.0001). Nematode-treated plants were shorter than plants that did not receive nematode treatment. Plants in non-sterile soil were taller than plants from sterile soil. The endophyte treatment did not affect plant height, number of leaves, leaf length and width (p > 0.05). Twenty-five other fungi were isolated from the root pieces, 11 genera among them were identified i.e. (i) Fusarium sp., (ii) Rhizoctonia sp., (iii) Trichoderma sp., (iv) Sclerotium sp., (v) Bipolaris sp., (vi) Curvularia sp., (vii) Mucor sp., (viii) Rhizopus sp., (ix) Papulaspora sp., (x) Pleurophragmium sp., and (xi) Trichocladium sp. At least fourteen of the microbes (that included ii, iii, vi, xi, viii, ix, x, xi) inhibited F. oxysporum V5w2; seven were inhibited by the endophyte among them Sclerotium sp., while Bipolaris, Mucor among other unidentified microbes showed no negative interactions. Among the Fusarium sp. that were identified, some inhibited F. oxysporum V5w2, others were inhibited while the remaining ones did not interact inhibitively with the endophyte (See Fig. 5.1).
100 Effects of soil sterilization on interactions between endophytic Fusarium.…
Figure 5.1. Top left: Outgrowths of Fusarium from surface-sterilized banana
roots in PDA. Top right: Growth inhibition test for Fusarium oxysporum V5w2 (center) and a root-invading microbes (4 outer). Bottom, 2nd from left: Fusarium compatible with F. oxysporum V5w2; Bottom, 3rd from left: Fusarium that is inhibitive to the endophyte.
The main effects of endophyte and nematode treatments on optical density of root extracts were significant (p < 0.0001) (Figure 5.2). Optical density of root extracts measured as absorbance at 320 nm for plants that were treated with the endophyte or with R. similis was lower than for control plants. The combination of nematode and endophyte treatment resulted in a similar optical density as any of the two treatments alone. Soil sterility did not affect the optical density of root extracts (p > 0.05).
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Discussion Under the conditions of the current study, we found that increased necrosis and percentage dead roots were apparent in all plants that were treated with R. similis resulting in a lower living root biomass. This confirms the pest potential of R. similis. In the present study, R. similis density and percentage dead roots were lower in banana plants that were grown in non-sterile soil than in sterile soil. Goes et al. (1995) reported similar observations in which soil populations of the nematode Telotylenchus ventralis that parasitizes Ammophila arenaria were reduced in non-sterile soil. It is likely that soil biota such as the root-invading microbes that we identified in samples from non-sterile soil help suppress R. similis and other nematodes. Further suppression of soil populations of R. similis in our experiment may have been a result of competition by the other identified plant parasitic nematodes (H. multicintus, Meloidogyne spp., P. goodeyi). Competitive interaction between R. similis and the plant parasitic nematodes previously mentioned was observed by Moens et al. (2006).
1
0.9 a Figure 5.2. Spectrophotometric analysis a 0.8 of root extracts from tissue culture 0.7 banana plants treated with nematode (R. 0.6 similis), endophyte (F. oxysporum b b b 0.5 V5w2) or both (nema+endo), or left b b 0.4 b untreated (control) and grown in sterile 0.3 or non-sterile soil, and supplied with
Optical densities (absorbance) at 320 nm at (absorbance) densities Optical 0.2 complete nutrient solution. Means with
0.1 the same letter not significantly different
0 (F test, LSmeans, p > 0.05). control nematode endophyte nema+endo
sterile soil non-sterile soil
102 Effects of soil sterilization on interactions between endophytic Fusarium.…
In the present study, banana plants grown in non-sterile soil exhibited better growth, had heavier roots and more leaves compared to those planted in sterile soil. This observation is similar to the finding of Smith and Smith (1981), that recorded improved growth of Trifolium sp. plants in non-sterile soil compared to sterile soil, attributed to infection by vesicular arbuscular mycorrhizal fungi (AMF). Similar reports on the improvement of plant growth through root promotion by rhizosphere microbes including AMF have been presented by Kothari et al. (1990) and Declerck et al. (2002). However, Evan et al. (2003) cited iron deficiency due to sterilizing soil in an autoclave as the cause of retarded growth of rape plants. We found that R. similis density, root damage and plant growth parameters were not different between nematode and nematode+endophyte treatments. We did not quantify the presence of F. oxysporum V5w2 in the experimental plants after being exposed to the experimental conditions. Thus, we have no information on the establishment of the endophyte in the inoculation treatments. However, root extracts from endophyte-inoculated plants had lower optical density than non-inoculated plants. Also the optical density of root extracts from nematode-treated plants was lower than that from the non-treated plants. Combined treatments of roots with the endophyte and R. similis resulted in an optical density that was not different from independent applications of the two treatments. This corroborates data on R. similis densities that are similar in nematode-treated plants that were inoculated with F. oxysporum V5w2 and those without the endophyte. Our
103 Chapter 5 data show that soil sterilization eliminates plant growth promoting microbes and the natural suppressive effect of soil on R. similis in banana plants.
Preparation of sterile nematode cultures on carrot discs (Elvis)
Acknowledgements This work was conducted under the management of the International Institute of Tropical Agriculture (IITA) in Uganda. The funds were provided by the German Ministry of Economic Cooperation and Development (BMZ) and Wageningen University. Technical support that was provided by Jane Luyiga, Phillip Abidrabo, Patrick Emudong, Elvis Mbiru, Fred Kato, Victoria Naluyange, Rose Khainza and Juliana Nakintu. Dr. Phillip Ragama advised on statistical analysis. We give credit to the Soil Chemistry Laboratory of Kawanda Agricultural Research Institute (KARI) for the training and analysis of soil samples. We express gratitude to the Conservation and Sustainable Management of Below-Ground Biodiversity (CSM-BGBD) project in Makerere University for the training they offered towards the identification of root invading microbes.
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Interactions between Radopholus similis and Fusarium oxysporum V5w2 in tissue culture banana plants under nitrogen-starvation and total nutrient starvation as affected by soil sterility
Assessment of harvested plants in the laboratory (Juliana and Nteza)
Dennis M.W. Ochieno · Marcel Dicke · Thomas Dubois · Danny Coyne · Piet J.A. van Asten · Arnold van Huis
Chapter 6
Abstract The potential of Fusarium oxysporum V5w2 for the control of Radopholus similis as affected by soil sterility was investigated in two concurrent experiments using potted banana plants (Musa spp.). Exp. 1 assessed the potential under N-starvation, by treating plants with a solution that contained all essential nutrients except N. All plants in Exp. 2 were subjected to total nutrient starvation by giving them only water. In both experiments, plants inoculated with R. similis had low root biomass, high percentage dead roots and necrosis. In Exp. 1, plants that were co-inoculated with R. similis and F. oxysporum V5w2 had lower percentage dead roots and lower nematode density compared to those treated with R. similis only; such differences were not recorded in Exp. 2. In Exp. 1, plants that were inoculated with R. similis had higher shoot dry weight, were taller with more leaves that were larger, compared to plants not inoculated with the nematode; such differences were not recorded in Exp. 2. In both experiments, plants that were grown in non-sterile soil had lower percentage dead roots, necrosis and density of R. similis compared to those from sterile soil. Also in both experiments, plants that were grown in non-sterile soil were shorter with lower biomass and smaller leaves compared to those from sterile soil. Our results indicate that sterilization eliminated R. similis- suppressive factors that may have existed in non-sterile soil, but promoted plant growth under the two nutrient starvation conditions. These results support the role of F. oxysporum V5w2 in the suppression of R. similis density and root damage in potted tissue culture banana plants growing under N-starvation, but not under total nutrient starvation.
106 Interactions between Radopholus similis and Fusarium oxysporum………...
Introduction The root burrowing nematode Radopholus similis is a serious pest of banana plants (Musa spp.) (Marin et al. 1998). The spread of R. similis in banana growing areas of the world and the crop losses that result from their infestation have raised global attention for the nematode as a quarantine pest (O'Bannon 1977; EPPO/CABI 1997). Infection of banana plants with R. similis is characterized by necrotic lesions and root death that leads to plant toppling (Marin et al. 1998; Gowen et al. 2005). Control of R. similis in banana plants has been very difficult (Bridge 2000), and crop losses of up to 51% have been associated with R. similis among other nematodes in the field (Speijer and Kajumba 2000). The use of pest-free planting materials that are produced by methods like tissue culture is a fundamental measure for minimizing R. similis among other infections in banana plants (Krikorian and Cronauer 1984; Vuylsteke 1998; Bridge 2000). Tissue culture banana plants are usually grown in sterilized soil to maintain their pest-free condition before they are taken to farms. However, tissue culture plantlets have been considered to be devoid of microbial agents that may offer protection against pests such as nematodes (Sikora et al. 2000). This has prompted research into introducing endophytic microbes in the plant as a biological control measure. In Uganda, research is being conducted on the use of microbial agents for endophytic control of nematode pests in banana plants (Dubois et al. 2006a). Among the microbes being tested for endophytic control of R. similis is the fungus Fusarium oxysporum V5w2 (Athman et al. 2007). This fungal strain is among a collection of microbes that were obtained from
107 Chapter 6 surface-sterilized banana roots in Uganda (Schuster et al. 1995; Paparu et al. 2009a). Research has been underway to investigate whether the biological control potential of F. oxysporum V5w2 is dependent on external conditions. Experiments investigating the effect of F. oxysporum V5w2 on tissue culture banana plants and R. similis have been conducted in pots using sterilized soil. Although sterilization eliminates background effects of soil biota, more information on the effect of such microbes may help in determining the performance of F. oxysporum strains under field conditions. Also, chemical depletion from pots without fertilizer application may cause deficiencies in nutrients such as nitrogen (N) in banana plants. Malnourished plants may exhibit changes in host suitability for the endophytes and nematodes, for example, in terms of nutrient suitability and expression of resistance. Soil microbes and nutrient deficiencies may affect the suppressiveness of F. oxysporum V5w2 against R. similis. The objectives of this study were to investigate the effects of F. oxysporum V5w2 and soil sterility on R. similis in potted banana plants grown under (1) N-starvation and (2) under total nutrient starvation.
Materials and methods Experimental design We conducted two concurrent experiments to investigate the biological control potential of F. oxysporum V5w2 against R. similis: under extreme N-starvation (Exp. 1), and under total nutrient starvation (Exp. 2). The treatments, viz. nematode vs. no nematode, endophyte vs. no endophyte,
108 Interactions between Radopholus similis and Fusarium oxysporum………...
sterile vs. non-sterile soil were studied in a full factorial experiment of eight treatments (2 × 2 × 2). The treatments, each having 15 plants, were repeated three times in each experiment. Plants in two of the replicates in both experiments were completely randomized in the greenhouse. In Exp. 1, all plants were supplied with water and an N-deficient nutrient solution. Those in Exp. 2 were treated with water only. N-deficient solution has been applied on plants to induce stresses related to N-starvation (Lancien et al. 1999), while giving them (deionized) water only results in total nutrient starvation (Ferreira and Davies 1987). One hundred days after the start of the experiment, data were collected on plant growth and nematode damage parameters, and for the presence of root-invading microbes.
Banana plants Tissue culture banana plants (genomic group AAA-EA, cv. Kibuzi) were obtained from IITA, Namulonge, Uganda. The plants had been micro- propagated by the shoot-tip culture technique in which plants are produced by multiplication of the meristematic tissue of banana corms (Vuylsteke 1998). The banana plants had been maintained for six weeks in nutrient solution (1 g/L, Polyfeed™, Haifa Chemicals, Israel), after which they were graded into four sizes and equally distributed to the eight treatment groups of 15 plants. Four groups were marked for inoculation with F. oxysporum V5w2 and the other four were controls. Initial fresh weights and number of functional roots were recorded.
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Fungal inoculum, nematodes and soil We used F. oxysporum strain V5w2 stored at IITA, which was originally isolated from banana roots and corms in Uganda (Schuster et al. 1995) and modified into a nit3 fungal strain by Paparu et al. (2009a). Inoculum was prepared by sprinkling the soil culture onto 90 mm diameter Petri dishes containing half-strength PDA (19 g/L, Sigma-Aldrich, Germany). Ten days later, the fungal spores and mycelia were scraped from the media into a 500 ml beaker containing sterile water. The spore mixture was sieved and homogenized, and then adjusted to 1.5 × 106 spores/mL. Radopholus similis were originally obtained from banana fields at Namulonge, IITA and the culture was maintained aseptically on carrot discs (Speijer and De Waele 1997). Nematodes for the experiments were supplied in sterile water suspension containing mixed stages of 250 R. similis/mL. Loamy soil was collected within a depth of 10 cm from a 5-year old unmulched banana field at Namulonge. The soil was sieved (5 mm aperture) before being thoroughly mixed. Soil samples were analyzed for chemical properties at the National Agricultural Research Organization (NARO), Kawanda, Uganda. Available P and exchangeable K were extracted using the Mehlich-3 method (Mehlich 1984). Phosphorus (P) in the extract was determined using the molybdenum blue colorimetric method and K using a flame photometer (Okalebo et al. 2002). Total N was analyzed by Kjeldahl oxidation and semi-micro Kjeldahl distillation (Bremner 1960). Organic matter (OM) was determined using the Walkley-Black method (Walkley 1947). Soil pH was analyzed using deionized water with a soil to water ratio of 1:2.5. The soil contained N (0.15 %), P (3.6 ppm), K (149 ppm i.e. 0.38
110 Interactions between Radopholus similis and Fusarium oxysporum………...
cmolc / kg), OM (2.4 %) and 5.1 pH. Part of the loamy soil was steam- sterilized at 100ºC for 1 h using an electrode steam conditioner (Marshall- Fowler, South Africa). Analysis of soil chemical properties was not done after sterilization.
Fungal inoculation, planting, and nematode infestation Plants marked for endophyte treatment were inoculated by the root-dipping technique (Paparu et al. 2006a), except that inoculation did not involve prior breaking of the root tips. The roots were immersed into a 1.5 L spore suspension contained in tubs (30 × 25 × 15 cm, LWH) for 4 h. Control plants were immersed in distilled water. The banana plants were grown in 2.5 L buckets with 6 holes at the base containing either sterile or non-sterile soil under screenhouse conditions (25±3ºC, 70-75 % RH, 12L: 12D photoperiod). Initial plant heights were recorded. The plants in both Exp. 1 and Exp. 2 were supplied with rain water (100 ml) daily. Plants in Exp. 1 were starved of N by supplying them with an N-deficient solution (100 ml) once every week. We formulated the N-deficient nutrient solution by omitting N from the chemical ingredients in the solution prepared by Murashige and Skoog (1962). One litre of the N-deficient solution contained 1401 mg KCl, 440 mg CaCl2.2H2O, 370 mg MgSO4.7H2O, 170 mg KH2PO4, 37.3 mg
Na2EDTA.2H2O, 27.8 mg FeSO4.7H2O, 6.2 mg H3BO3, 22.3 mg
MnSO4.4H2O, 8.6 mg ZnSO4.7H2O, 0.83 mg KI, 0.25 mg Na2MoO4.2H2O,
0.025 mg CuSO4.5H2O and 0.025 mg CoCl2.6H2O. Those for the experiment on interactions under total nutrient starvation (Exp. 2) were not
111 Chapter 6 treated with any fertilizer but grew on nutrients that were already in the soil. Twenty days after planting, three holes (5 cm deep) were made into the soil around the base of each plant, by using a disinfected stick. Each plant was inoculated with ~500 female R. similis in 2 ml of the nematode suspension, distributed across the three holes before covering them with soil. Initial plant growth-related parameters in both experiments did not vary between the treatments (F test, p > 0.05). Values for initial parameters in Exp. 1 were fresh weight (8.5 ± 0.24 g), plant height (5.3 ± 0.15 cm), number of leaves (3.3 ± 0.05), leaf length (12.4 ± 0.19 cm), leaf width (4.8 ± 0.08 cm) and number of functional roots (3.6 ± 0.07) (n = 340, mean ± SE). Values for initial parameters in Exp. 2 were fresh weight (8.4 ± 0.23 g), plant height (5.3 ± 0.14 cm), number of leaves (3.3 ± 0.04), leaf length (12.5 ± 0.18 cm), leaf width (4.8 ± 0.08 cm) and number of functional roots (3.6 ± 0.06) (n = 340, mean ± SE).
Data collection At 100 days after nematode inoculation, plant height, number of leaves, length and width of youngest open leaf were recorded. Plants were harvested and the numbers of dead and healthy roots were recorded and used to calculate the percentage dead roots. The shoots and roots were detached and their fresh weights recorded. Root and shoot biomasses were combined for total plant biomass. The shoots were oven-dried at 70ºC for a discontinuous period of 14 days to determine their dry weight. The roots were preserved at 4ºC awaiting the extraction of nematodes and root- invading microbes.
112 Interactions between Radopholus similis and Fusarium oxysporum………...
Five roots per plant were randomly selected for assessment of necrosis caused by R. similis, and for estimation of nematode populations based on the methods described by Speijer and DeWaele (1997) and Brooks (2004). Three other roots were selected per plant for extraction of root- invading microbes. The roots were surface-sterilized by dipping them into 96% ethanol followed by flaming to ensure that only microbes within the tissues remained alive. Each root was then cut into six pieces that were plated on 60 mm glass Petri dishes containing PDA that contained KClO3 (30 g/L). The microbes were allowed to grow within 7 days. Root-invading microbes growing from the root pieces in the two experiments were sub- cultured and identified using keys at the Soil Microbiology Laboratory (Makerere University, Uganda).
Statistical analysis Data were analyzed using SAS 9.1 software (SAS Institute Inc, 2001). Diagnostic check for normality was conducted using proc univariate. Since pre-analysis of data from the three replicates yielded similar results in both Exp. 1 and Exp. 2, they were all treated as completely randomized. Proc transreg was used to find appropriate Box-Cox transformations, which included generation of suitable powers or lambda (λ) for data (Y) that required transformation. In Exp. 1, lambda for root biomass was estimated as 0.75 hence the transformation became Y0.75. Lambda was 1.25 for total biomass, shoot fresh weight and leaf length. For plant height and leaf width λ was 1.5; and 0.5 for number of leaves and shoot dry weight.
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In Exp. 2, lambda for shoot dry weight was estimated as 0.5 hence the transformation became Y0.5. Lambda was 1.25 for leaf length, and 1.5 for the width. Lambda was 0.75 for plant height, root weight, shoot fresh weight and total biomass. Untransformed data were used for comparison of number of leaves. In both Exp. 1 and Exp. 2, percentage dead roots and percentage necrosis (x) were arcsine√(x/100) transformed, while untransformed data were used for nematode densities. Proc glm was used for three-way factorial analyses of variance (ANOVA) among the treatments. Mean separation was done by t-tests (LSD) when ANOVA was significant.
Results Experiment 1: Interactions under N-starvation (Table 6.1) All plants expressed symptoms that may be linked to N-starvation in the form of being stunted with yellow leaves that had pinkish veins. The leaves dried from the margins towards the midribs, and wilted from the lower older ones, towards the upper young ones. Dead roots, necrosis and R. similis were mainly found in nematode- treated plants. Nematode-treated plants in sterile soil had higher percentage dead roots than those from non-sterile soil, both in the presence and in the absence of the endophyte. Among nematode-treated plants in non-sterile soil, endophyte-treated plants had a lower percentage dead roots than those without the endophyte. In contrast, among nematode-treated plants in sterile soil, there was no difference in percentage dead roots between endophyte- treated plants and those without the endophyte. Nematode-treated plants in
114 Interactions between Radopholus similis and Fusarium oxysporum………...
sterile soil had a higher percentage root necrosis and higher density of R. similis than those from non-sterile soil. Plants from sterile soil that were not treated with nematodes had a higher root biomass compared to those from the remaining treatments, which had similar root biomass. Plants from sterile soil had higher total biomass than those from non-sterile soil. In sterile soil, nematode-treated plants had lower total biomass than those without nematodes. However, in non-sterile soil, total plant biomass did not vary between nematode-treated and nematode-free plants. Shoot dry weight, plant height and size of leaves had higher values in nematode-treated plants than in those without nematodes. Endophyte- treated plants that were inoculated with R. similis had more leaves than those that were not treated with the nematode. In non-sterile soil, plants that were treated with R. similis had more leaves than those without the nematode. Plants that were treated with R. similis appeared to have greener leaves than those without the nematode. Shoot fresh and dry weights, plant height, length and width of leaves were greater in plants from sterile than those from non-sterile soil. Root-invading microbes that were identified mainly from our non- sterile soil samples in both experiments included Fusarium spp., Rhizoctonia spp., Trichoderma spp., Sclerotium spp., Bipolaris spp., Curvularia spp., Mucor spp., Rhizopus spp., Papulaspora spp., Pleurophragmium spp. and Trichocladium spp.
115 Chapter 6
Experiment 2: Interactions under total nutrient starvation (Table 6.2) All plants were stunted with a complex of symptoms related to total nutrient starvation, although yellowing and drying of leaves were most prominent. Radopholus similis density, percentage dead roots and necrosis were higher in nematode-treated plants in sterile soil than in non-sterile soil. In nematode-free plants, root necrosis was higher in non-sterile than in sterile soil, but percentage dead roots and R. similis density did not vary between the soils. In sterile soil, nematode-treated plants had lower total biomass than those without nematodes. However, in non-sterile soil, total plant biomass was similar between nematode-treated plants and those without nematodes. Plants in sterile soil had more total biomass than those from non-sterile soil. Observations on root biomass had similarities with those of total plant biomass. Shoot fresh weight was lower in plants that were treated with R. similis than in those that were not treated with the nematode; and was significantly higher in sterile than in non-sterile soil. Shoot dry weight was significantly lower in plants from non-sterile than from sterile soil. Plants grown in non-sterile soil were shorter than those from sterile soil. Leaves of nematode-treated plants were smaller than those from plants that did not receive nematode treatment. Plants from sterile soil produced larger leaves than those from non-sterile soil. The number of leaves did not vary between treatments.
116 Interactions between Radopholus similis and Fusarium oxysporum………...
e 326 e 29.0 b b 29.0 b 13.3 31.2 14.5 33.1 a 33.1 a 15.0 32.4 15.0 30.6 13.7 30.1 13.5 326 e 3.8 a 3.9 3.8 Leaves ab 3.8 3.5 b ab 3.8 328 e 25.7 a 25.7 b 24.0 a 3.9 b 3.7 a 31.8 b 30.3 a 14.7 b 13.6 Number Length Width 328 fwt e 180.4 187.1 V5w2 and soil sterility on root damage, damage, root on sterility soil and V5w2 Total dwt 326 e 13.9 a 13.9 12.5 b Shoot 326 fwt e 133.9 127.9 Fusarium oxysporum Shoot fwt 326 e Biomass Height Root 54.0 51.6 129.6 132.3 13.2 13.2 183.5 183.9 24.9 24.8 3.7 3.8 30.9 31.2 14.1 14.3 gram gram gram gram cm number cm cm -100g 326 e count R. similis 10372 b a 14773 322 , the fungal endophyte e 45.9 20320 49.1 132.3 13.5 181.4 25.3 24.2 23.7 46.0 2.1 28195 1.7 60 43.6 1185 58.9 135.7 59.5 126.8 14.3 129.0 12.9 12.1 179.2 185.7 26.2 188.5 24.5 23.4 Means Means 326 e 27.4 b 10.9 b 20.0b b 6855 46.0 b 106.1 b 10.8 b 152.1 b 22.6 b
Death Necrosis a 28.8 b 1.1 a 45.9 b 1.9 a 24187 14.3b b b 619 46.4 a 15.7 a 59.2 a 19.0 a 27.7 a 18237 a 59.5 30.3 a a 155.2 c1.0 a 15.6 c1.3 a 214.6 a 27.0 percent percent
Radopholus similis 326 trt 1 31.4*** 4.5* 35.4*** 33.3*** 1 166.0*** 71.1*** 5.1* 125.6*** 59.6*** 0.6 0 0.3 65.9*** 65.2*** 0.2 0 1.7 0 0.1 0.6 1.2 1.2 df Root damage 1 5.8* 0.1 1 5.9* 4.0* 1.0 0.6 0.7 3.5 0 1.9 0 0 0 1.3 1.3 0.3 0.6 3.1 0.6 4.1* 3.0 3.0 1 978.4*** 1237.3*** 151.2*** 29.4*** 3.1 6.5* 1.4 9.9* 4.8* 9.6** 9.9** F values values F
7 50.2 43.4 139.7 40.2 32.5 20.7 34.0 30.1 11.0 18.3 21.7
1 69.9*** 69.8*** 32.4*** 22.2*** 1.8 0.4 9.1** 0.9 9.5** 1.4 1.1 1 18.0*** 3.1 0.1 4.1* 1.0 0.1 2.2 0.1 0.2 0.2 0.2
15.0 23.9 12546 52.8 130.9 13.2 183.7 24.9 3.8 31.1 14.2
Fo Rs Fo Fo
) ) end nem Effects of the nematode of Effects the (Rs (Fo
× S Fo Fo × S × × S × Nematode Non-sterile Grand mean Two-way interactions Soil sterility (S) Two-way Rs Rs Source of variation variation of Source effectsMain Nematode Endophyte Nematode non Endophyte Soil non Sterile nematode No non non
Three-way interactions Fo Three-way Rs c.v. Total (df) nematode density, plant biomass, height, number and size of leaves in tissue culture banana plants grown under N-starvation. N-starvation. under grown plants banana culture tissue in leaves of Table size and 6.1. number height, biomass, plant density, nematode
117 Chapter 6 33.5 15.3 30.1 14.2 32.7 14.7 27.9 12.5 3.7 ab ab 3.7 4.0 a 4.0 ab 3.8 3.5 b 27.6 23.9 26.5 21.4 dryweight; Asterisks indicate significant effect dwt 203.0 b 157.4 c 226.1 a 146.7 c Fresh weight and and weight Fresh fwt 155.4 16.3 158.8 111.9 16.3 112.5 10.9 155.8 12.2 206.6 153.9 15.8 162.7 96.3 28.0 14.2 151.9 103.4 23.4 9.9 228.4 3.7 24.4 10.1 223.7 4.1 27.2 140.9 34.0 4.0 25.8 152.4 29.3 3.6 21.7 15.5 30.9 4.0 21.1 13.7 33.3 3.5 14.6 32.0 3.6 14.8 27.8 14.5 28.0 12.5 12.5 112.2 11.5 154.9 15.0 99.9 10.0 152.2 16.2 199.7 27.1 3.8 33.1 15.2 47.8 b b 47.8 b 50.9 b 39.4 a 72.5 a 69.8 b 44.6 b 49.0 47.5 b b 47.5 degreesError of freedom; e Treatment and 53.2 33.5 40432 38.5 9688 0.1 16541 0.1 31 4.3 1713 3.4 90 644 29.0 15733 60.0 154.0 16.0 214.0 27.2 3.7 33.2 15.0 19.0 26.3 4947 20834 47.8 58.9 104.2 156.3 10.4 15.2 151.9 215.2 22.5 26.9 3.8 3.9 28.5 33.0 13.1 15.0 21.0 8786 57.8 44.2 30705 108.0 11.1 152.2 22.7 3.8 29.5 13.6 trt V5w2; 37.5 a 37.5 a 55.5 a 35393 b 47.6 35.6 a c 15.1 25.0 b 0 d 1.3 d 2.1 d 1.2 d 20.0 b c 0.6 36.0b d 0.1 13114b c 872 45.2 b a 71.2 1.7 c 1.7 c 3.8 19.6 a c 363 b 46.8 8.7 b 18.2 a 13.1 a 39.3 a
Fo Fo Fo non
Fo 0.05); Means for significant effects are indicatedfont, bold in thosewith the same letternot significantly are different0.05) (p > ≤ Fusarium oxysporum oxysporum Fusarium end 0.01, *p Non-sterile non
≤ 0.001, **p ≤ Radopholus similis; Nematode Sterile Non-sterile Non-sterile Non-sterile Sterile non No nematode Sterile Non-sterile nem Sterile non (***p No nematode Non-sterile Sterile Endophyte Sterile Non-sterile No endophyte Non-sterile Sterile Nematode Sterile
118 Interactions between Radopholus similis and Fusarium oxysporum………...
e 321 e 26.8 b 27.8 a 12.2b 12.9a 29.7 a 24.6 b 13.6a 11.4b 321 . e 3.8 3.7 321 e Height Leaves 22.1 22.8 3.7 3.8 Number Length Width 321 e fwt fwt 131.5 b 154.9 a V5w2 and soil sterility on root damage, nematode nematode damage, root on sterility soil and V5w2 Total dwt 328 e 11.4 12.4 Shoot fwt 326 e Fusariumoxysporum Shoot 328 e fwt Biomass Biomass Root gram gram gram gram cm count cm cm 328 e -100 g count R. similis similis R. 18606a 54.0 4437 a b 31.6 b 124.1 a 73.8 b 14.7 a 8.9 b 178.0 a 105.4 b 24.9a 19.7 b , the fungal, the endophyte 322 e percent percent Necrosis Necrosis 325 e Radopholus sim ilis 27.5 29.3 Means Means Death 47.5 a 9.4 b 47.2 a 6.5 b 22572a 29.7 36.1 b 631 b 27.6 50.5 a 27.5 95.4 b 26.6 104.4 a 12005 11734 43.3 47.2 43.1 47.8 101.1 47.9 12.0 98.6 46.6 6.7 11.9 23612 7.1 11.9 21544 6.0 144.4 35.7 675 141.7 36.5 580 22.7 50.9 96.8 22.2 50.0 94.0 3.7 105.4 11.6 3.8 103.4 11.2 132.5 12.1 27.6 12.6 130.5 27.0 156.4 12.7 22.4 153.3 12.4 21.7 3.7 23.0 22.7 3.7 3.7 27.1 3.9 26.4 12.4 28.0 27.6 12.0 13.0 12.8 28.7 27.1 11874 43.2 99.9 11.9 143.1 22.5 3.7 27.3 12.6 328 trt 1 1.0 0 33.7*** 108.5*** 209.6*** 153.1*** 180.3*** 76.4*** 1.2 108.3*** 82.3*** F values values F
1 2.2 1.5 0.2 0.1 0.3 0 0 0.8 2.4 1.6 1.8 1 434.6*** 769.2*** 78.6*** 44.5*** 6.3* 3.2 17.5*** 1.4 1.2 4.7* 8.9** 1 1 3.3 71.3*** 113.4*** 0.4 34.8*** 13.6*** 0.2 1.4 0 3.0 0.1 4.7* 5.6* 0.4 0.9 0 3.3 0.1 2.4 3.5 0.2 0.3 1 3.3 0.1 0.5 5.7* 2.5 0.3 3.8 0.1 0.4 0.4 0 df df damage Root 7 58.9 42.4 189.0 35.0 24.6 19.3 26.7 18.4 18.9 19.6 24.2 1 3.8 0.7 0.1 13.1*** 2.9 0.1 6.4* 0.8 0.1 1.0 0.1
Rs Fo non Sterile Non-sterile Fo non 29.9 Fo non 24.6 ) ) end nem
(Fo of Effects the nematode (Rs
× S
percent Soil (S) sterility Source of variation Main effects Nematode Endophyte Nematode non Endophyte Soil Nematode No nematode Grand mean Two-way interactions Rs × Fo Rs × S Fo × S
Three-way interactions Three-way Rs × Fo c.v. Total (df) Table 6.2. Table density,plant biomass, height, number andsize ofleaves in tissue culturebanana plants grown under total nutrient starvation
119 Chapter 6 p 0.05) > 3.7 28.8 13.0 3.7 24.6 11.3 3.9 30.6 14.1 3.7 24.6 11.6 23.7 3.7 28.8 12.8 20.6 3.7 25.4 11.5 19.5 3.7 23.8 11.1 26.5 3.7 30.9 14.2 25.5 4.0 30.3 13.9 24.0 3.7 28.8 13.3 19.2 3.6 24.9 11.7 19.6 3.9 24.7 11.6 dryweight; Asterisks indicate significant effect dwt 160.1 b 23.9 a 161.1 b 101.6 c 20.1 b 104.7 c 196.0 a 26.0 a 98.4 c 98.4 109.5 c 19.3 b 208.8 a 183.1 ab 159.1 b 99.9 c 99.9 122.5 c Fresh weight and and weight Fresh fwt 117.4 13.7 116.4 13.4 72.4 9.0 74.1 9.0 130.8 15.6 70.6 9.0 75.4 8.7 137.1 15.4 124.4 15.9 127.8 72.8 14.7 120.4 8.8 74.9 184.0 14.6 102.3 118.5 8.9 25.3 172.1 19.9 14.1 108.6 3.7 24.6 3.6 19.4 29.8 3.8 25.1 3.8 13.7 29.6 11.6 24.1 13.4 11.3 71.4 8.7 81.3 9.1 44.7 b 30.5 bc 27.8 c 27.8 71.7 a 58.7 a 56.2 a 29.6 b 51.7 a 33.7 b 40.6 b 28.6 bc 41.3 b Error degrees of freedom; of freedom; degrees Error e Treatment and trt trt V5w2; 54.2 a 57.5a 36098 a 42.7 b 40.6 b 36.2b 7847 b c 29.2 0.9 d 1.1 d 483c 65.2 a 18.7 c 18.7 c 12.6 27.2 796c 34.2 bc 30.0 17949 27.8 28.6 19288 23.1 36.3 10193 0.1 1.2 116 36.3 58.8 35782 11.9 13.4 1261 Fo Fo Fo Fo 0.05); Means 0.05); for significant effects indicated are font, bold in those withnot same the letter significantly are different( ≤ Fusarium oxysporum oxysporum Fusarium end 0.01, *p ≤ Sterile no 36.8 56.3 36414 Non-sterile no 22.9 36.1 5560 Sterile no 0.3 1.0 877 Non-sterile Non-sterile 32.4 24.8 27.4 5615 24.4 3146 Non-sterile no 4.8 11.0 177 0.001, **p ≤ Radopholus similis; Sterile Nematode nem (***p Non-sterile Non-sterile No nematode Sterile
Non-sterile No nematode Sterile Endophyte Sterile
No endophyte Sterile Sterile Nematode Non-sterile
120 Interactions between Radopholus similis and Fusarium oxysporum………...
Discussion Economic losses resulting from root damage by R. similis in banana plants have prompted research on the use of non-pathogenic F. oxysporum V5w2 for endophytic control of the nematode pest. In both Exp. 1 and Exp. 2, plants that were treated with R. similis had a high percentage of dead roots and necrosis. Root biomass was low as a consequence of damage by R. similis. Such damage confirms the detrimental effects of R. similis on banana plants (Speijer and De Waele 1997; Viaene et al. 2003). In Exp. 1 that was conducted under the supply of N-deficient solution, inoculation of banana plants with F. oxysporum V5w2 resulted in the suppression of R. similis density and lower root death than in plants without the endophyte. However, root necrosis was not different between plants that were co- inoculated with R. similis and F. oxysporum V5w2, and those inoculated with the nematode only. In contrast to Exp. 1, we did not record an effect of the fungus F. oxysporum V5w2 for the assessed parameters when plants were grown under total nutrient starvation in Exp. 2. The mechanisms involved in the suppression of banana root damage and R. similis density by F. oxysporum V5w2 under the supply of N- deficient solution in Exp. 1 remain to be elucidated. We found Fusarium spp. among the root-invading microbes, but we could not verify whether they were the inoculated F. oxysporum V5w2. It would be interesting to investigate whether the suppression of R. similis by F. oxysporum V5w2 under the supply of N-deficient solution results from endophytic or from rhizosphere colonization by the fungus in potted banana plants.
121 Chapter 6
Growth was constrained by R. similis in plants that were grown under total nutrient starvation in Exp. 2. In contrast, plants that were treated with R. similis had improved growth when exposed to an N-deficient solution in Exp. 1. Growth improvement was reflected in slightly higher values in shoot dry weight, plant height, number and size of leaves in nematode-infected plants than in control plants. Plants that were inoculated with R. similis also appeared to be greener than those without the nematode in Exp. 1. Other authors have reported improvement of root biomass by plant-feeding nematodes (Bardgett et al. 2001; Moens et al. 2006). Enhancement of plant growth by plant-feeding nematodes has been associated with improvement of soil N content through processes such as excretion of ammonia, defaecation and decomposition of their bodies (Verschoor 2002). However, this occurred in the extreme conditions under which Exp. 1 was conducted. In both Exp. 1 and Exp. 2, nematode density, percentage dead roots and necrosis were lower in non-sterile than in sterile soil. Such effects may be attributed to the presence of nematode-suppressive microbes in non- sterile soil. Some fungal genera like Fusarium and Trichoderma that we recorded among the root-invading microbes may have included species that have been mentioned to have nematode suppressive effects (Sikora et al. 2008). In both Exp. 1 and 2, plant biomass, height and leaf size had lower values in non-sterile than in sterile soil. Microbial demand for scarce nutrients especially nitrogen in non-sterile soil may have constrained plant growth. Growth may also have been better in sterile than in non-sterile soil
122 Interactions between Radopholus similis and Fusarium oxysporum………...
due to increased availability of nutrients caused by sterilization (Troelstra et al. 2001). Our results indicate that sterilization eliminated R. similis- suppressive factors that may have existed in non-sterile soil, but promoted plant growth under the two nutrient starvation conditions. These results support the role of F. oxysporum V5w2 in the suppression of R. similis density and root damage in potted tissue culture banana plants growing under N-starvation, but not under total nutrient starvation.
Replenishing nutrient solution for tissue culture banana plantlets (Ssebaggala)
Acknowledgements This work was conducted under the management of the International Institute of Tropical Agriculture (IITA) in Uganda. The funds were provided by the German Ministry of Economic Cooperation and Development (BMZ) and Wageningen University. Technical support that was provided by Jane Luyiga, Phillip Abidrabo, Patrick Emudong, Elvis Mbiru, Fred Kato, Victoria Naluyange, Rose Khainza and Juliana Nakintu. Dr. Phillip Ragama advised on statistical analysis. We give credit to the Soil Chemistry Laboratory of Kawanda Agricultural Research Institute (KARI) for the training and analysis of soil samples.
123
Chapter 7
Effects of endophytic Fusarium oxysporum V5w2 on the root burrowing nematode Radopholus similis in tissue culture banana plants under phosphorus and potassium deficiencies as affected by soil sterility
Assessment of roots for nematode damage (Dennis and Nteza)
Dennis M.W. Ochieno · Marcel Dicke · Thomas Dubois · Danny Coyne · Piet J.A. van Asten · Arnold van Huis
Chapter 7
Abstract Nutrient depletion may affect the performance of microbial biological control agents in the rhizosphere. The potential of Fusarium oxysporum V5w2 for the control of Radopholus similis in banana roots was investigated under phosphorus (P) and potassium (K) deficiencies, both in sterile and non-sterile soil. Plants were grown in pots and treated with nutrient solutions that contained all essential nutrients for banana growth, but with P and K omitted in two separate experiments. In both experiments, nematode- treated plants had higher levels of root necrosis, were shorter and had lower biomass than nematode-free ones. For all the investigated parameters, in both experiments, there was no indication that F. oxysporum V5w2 alleviated the negative effects of R. similis. To the contrary, under P- deficiency, roots co-inoculated with the nematode and F. oxysporum V5w2 had a higher density of R. similis than those inoculated with the nematode only. Root biomass was lower in plants that were inoculated with F. oxysporum V5w2 than those without the endophyte in plants under K- deficiency. Plants that were inoculated with F. oxysporum V5w2, and grown in sterile soil and under K-deficiency had fewer leaves than those under these conditions without exposure to the endophyte. In both experiments, nematode-treated plants in non-sterile soil had a lower percentage of dead roots, lower R. similis densities, greater root biomass and more leaves, compared to those from sterile soil. Leaves of plants from sterile soil were fewer and smaller than those from non-sterile soil. In these experiments, R. similis damages were lower and plant growth better in non-sterile than in sterile soil. The experiments do not provide evidence for R. similis control
126 Effects of endophytic Fusarium oxysporum V5w2 on the root burrowing…...
by F. oxysporum V5w2 in banana plants. Rather, F. oxysporum V5w2 enhanced nematode colonization in banana roots that were treated with P- deficient nutrient solution; and lowered root biomass and number of leaves in those that received K-deficient solution.
Introduction The root burrowing nematode, Radopholus similis (Cobb) Thorne, is a major pest of banana plantations (Musa spp.) in Eastern and Central Africa. The worldwide dissemination of R. similis has led to its classification as a quarantine pest (O'Bannon 1977; EPPO/CABI 1997). Root damage by this pest interferes with water and nutrient uptake in affected plants, reducing growth and weakening plant anchorage following necrosis and death of roots (Gowen et al. 2005). Banana crop losses up to 51% have been associated with R. similis among other nematodes in experimental fields (Speijer and Kajumba 2000). The use of pest-free planting material is a primary measure for the management of R. similis and other nematodes. Due to the high demand for banana products and an increasing pest and disease pressure (Gold et al. 2002), tissue culture has become an important tool for rapid multiplication of pest-free planting material (Vuylsteke 1998). Unfortunately, strict aseptic techniques employed in tissue culture multiplication do not only eliminate pathogens, but also beneficial endophytic microbes that offer protection to the crop against pests (Sikora et al. 2000). Tissue culture banana plants may therefore be vulnerable to infestation by pests in the field (Sikora et al. 2000). Protection of tissue culture banana plants through the introduction of
127 Chapter 7 pest-inhibiting microbes is an option for nematode control (Sikora et al. 2000; Dubois et al. 2006a). For example, Fusarium oxysporum V5w2 has been shown to suppress R. similis in banana roots (Vu et al. 2006; Athman et al. 2007). However, to understand whether pest-suppressiveness of F. oxysporum V5w2 is dependent on environmental factors, studies on the biological control potential of the endophyte are still necessary under different conditions. Pot trials that have investigated the control of R. similis by F. oxysporum V5w2 have been conducted using sterilized soil (e.g. Athman et al. 2007). Due to the elimination of microbes, experiments that are conducted using sterilized soil may not elucidate the biotic interactions that occur under field conditions (Troelstra et al. 2001). Sterilization also changes the chemical and physical properties of the soil (Smith and Smith 1981; De Deyn et al. 2004). For instance, the concentration of phosphorus has been shown to increase in sterilized soils (Serrasolsas and Khanna 1995; Anderson and Magdoff 2005). In pot experiments, the depletion of nutrients from the rhizosphere may contribute to variation in the biological control potential of root-invading microbes like F. oxysporum V5w2. Experimental results may therefore differ due to variation in nutrient concentrations. Such variations may be controlled when plant-nematode-microbe interactions are studied under uniform nutrient conditions. The objective of this work was to investigate the biological control potential of F. oxysporum V5w2 against R. similis in tissue culture banana plants, between sterile and non-sterile soil, under conditions of phosphorus or potassium deficiency.
128 Effects of endophytic Fusarium oxysporum V5w2 on the root burrowing…...
Materials and methods Experimental design Two separate experiments were conducted to investigate the interactions between R. similis and F. oxysporum V5w2 under phosphorus (P) and under potassium (K) deficiencies respectively. The treatments in both experiments, viz. nematode vs. no nematode, endophyte vs. no endophyte, sterile vs. non- sterile soil were studied in a full factorial experiment (2 × 2 × 2). This resulted in 8 treatments i.e. control, nematode, endophyte, and nematode+endophyte, each having been conducted in sterile and non-sterile soil. The treatments, each having 15 plants, were replicated thrice over time. Plants in two replicates in each experiment were completely randomized in the screen house. All plants in the phosphorus deficiency experiment were starved of P by supplying them with P-deficient nutrient solution, while those in the potassium deficiency experiment were starved of K by treating them with a K-deficient solution. One hundred days after the start of the experiment, data were collected on plant parameters that included (i) biomass (root, shoot, total), (ii) percentage dead roots, (iii) root necrosis, (iv) R. similis densities in roots, (v) plant height, (vi) number of standing functional leaves, (vii) leaf size (length and width).
Banana plants Tissue culture banana plants (genomic group AAA-EA, cv. Kibuzi) were obtained from the International Institute of Tropical Agriculture (IITA), Namulonge, Uganda. The plants had been micropropagated by the shoot-tip culture technique in which plantlets are produced by multiplication of the
129 Chapter 7 meristematic tissue of banana corms (Vuylsteke 1998). The banana plants had been maintained for six weeks in complete nutrient solution (1 g/L, Polyfeed™, Haifa Chemicals, Israel), after which they were graded into four sizes and distributed to eight groups of 15 plants. Four groups were marked for inoculation with F. oxysporum V5w2 and the other four were unmarked. Initial fresh weights and number of functional roots of the plants were recorded.
Fungal inoculum and nematodes Fusarium oxysporum V5w2 was originally isolated from banana roots in Uganda by Schuster et al. (1995) and modified into a nit-3 fungal strain by Paparu et al. (2009a). The endophyte culture comprised of sterilized soil medium stored in glass tubes at 4ºC in IITA, Uganda. Inoculum was prepared by sprinkling the soil culture onto 90 mm diameter Petri dishes containing half-strength potato dextrose agar (PDA, 19 g/L, Sigma-Aldrich, Germany). Ten days later, the fungal spores and mycelia were scraped from the media into a 500 mL beaker containing sterile water. The spore mixture was sieved, homogenized and adjusted to ~1.5 × 106 spores/mL. Radopholus similis were originally obtained from banana fields at Namulonge, IITA and maintained aseptically on carrot discs (Speijer and De Waele 1997). Nematodes were supplied in sterile water suspension containing mixed stages of ~250 R. similis/mL.
130 Effects of endophytic Fusarium oxysporum V5w2 on the root burrowing…...
Soil Loamy top soil (0-10cm) was collected from a bare 5-year old banana field at Namulonge. The soil at this site has been classified as isohyperthermic Rhodic Kandiudalf (USDA taxonomy) (McIntyre et al. 2000). The soil was sieved (5 mm aperture) and thoroughly mixed. Soil samples were analyzed for chemical properties at the National Agricultural Research Organization (NARO), Kawanda, Uganda. Available P and exchangeable K were extracted using the Mehlich-3 method (Mehlich 1984). Phosphorus in the extract was determined using the molybdenum blue colorimetric method and K using a flame photometer (Okalebo et al. 2002). Total N was analyzed by Kjeldahl oxidation and semi-micro Kjeldahl distillation (Bremner 1960). Organic matter (OM) was determined using the Walkley- Black method (Walkley 1947). Soil pH was analyzed using deionized water with a soil to water ratio of 1:2.5. The soil contained N (0.15 %), P (3.6 ppm), K (0.38 cmolc / kg), OM (2.4 %) and a pH of 5.1. Part of the loamy soil was steam-sterilized at 100ºC for 1 h using an electrode steam conditioner (Model ESC40, Marshall-Fowler, South Africa). Analysis of soil chemical properties was not done after sterilization.
Fungal inoculation, planting, and nematode infestation Plants marked for endophyte treatment were inoculated by the root-dipping technique (Paparu et al. 2006a), except that root tips were not broken. The roots were immersed into a 1.5 L spore suspension contained in tubs (30 × 25 × 15 cm, length × width × height) for 4 h. Control plants were immersed in distilled water.
131 Chapter 7
The banana plants were grown in 2.5 L buckets containing either sterile or non-sterile soil under screenhouse conditions (25 ± 3ºC, 70-75 % RH, 12L:12D photoperiod). The buckets were perforated at their bases with 6 holes to prevent possible water logging. Initial plant height, and length and width of the youngest open leaf were recorded as described by Akello et al. (2007). The plants were supplied with rain water (100 mL) daily and a weekly supply of nutrient solutions (100 mL) that were deficient in P and K respectively. We formulated the P- and K-deficient nutrient solutions by omitting P or K from the chemical ingredients in the complete solution formulated by Murashige and Skoog (1962). One litre of P-deficient nutrient solution contained 1650 mg NH4NO3, 1900 mg KNO3, 440 mg CaCl2.2H2O,
370 mg MgSO4.7H2O, 93 mg KCl, 37.3 mg Na2EDTA.2H2O, 27.8 mg
FeSO4.7H2O, 6.2 mg H3BO3, 22.3 mg MnSO4.4H2O, 8.6 mg ZnSO4.7H2O,
0.83 mg KI, 0.25 mg Na2MoO4.2H2O, 0.025 mg CuSO4.5H2O and 0.025 mg
CoCl2.6H2O. The K-deficient solution differed from the P-deficient one by not containing KNO3 and KCl, but having 171 mg of NaH2PO4.H2O (a negligible amount of K was retained in the form of KI due to unavailability of alternative source of iodine [I]). Twenty days after planting, three holes (5 cm deep) were made into the soil around the base of each plant using a disinfected stick. Each plant was inoculated with ~500 mixed stages of R. similis in 2 mL of the nematode suspension distributed across the three holes before covering them with soil. In the P-deficiency experiment, initial plant growth parameters did not vary among the treatments, and they included fresh weight (6.9 ± 0.20 g), plant height (5.5 ± 0.10 cm), number of leaves (3.7 ± 0.04), leaf length
132 Effects of endophytic Fusarium oxysporum V5w2 on the root burrowing…...
(11.2 ± 0.20 cm), leaf width (4.1 ± 0.10 cm) and number of functional roots (3.6 ± 0.08) (n = 350, mean ± SE, F test, p > 0.05). In the K-deficiency experiment, initial plant growth parameters also did not vary between the treatments, and they included fresh weight (8.1 ± 0.20 g), plant height (5.6 ± 0.12 cm), number of leaves (3.3 ± 0.03), leaf length (12.7 ± 0.17 cm), leaf width (4.1 ± 0.10 cm) and number of functional roots (4.9 ± 0.08) (n = 376, mean ± SE, F test, p > 0.05).
Data collection At 100 days after nematode inoculation, plant growth parameters, i.e. height, number of leaves, length and width of youngest open leaf were recorded. Plants were harvested and the number of dead and healthy roots were recorded and used to calculate the percentage dead roots. The shoots and roots were detached and their fresh weights recorded. Root and shoot biomasses were combined to obtain total fresh weight. The shoots were oven-dried at 70ºC for a discontinuous period of 14 days to determine their dry weight. The fresh roots were maintained at 4ºC within three days of harvesting. Five roots per plant were randomly selected for assessment of necrosis, and for estimation of nematode density. Root necrosis assessment and quantification of nematode densities was undertaken based on the methods of Speijer and De Waele (1997). Each of the five roots selected per plant for necrosis assessment was cut into 10 cm length. The pieces were then split longitudinally and one half selected from each of them. The cumulative length of necrotic lesions of cortical tissue on each 10 cm side of the stele was recorded as a fraction of the total 20 cm length per root. The
133 Chapter 7 sum length of the necrotic tissue for five roots per plant was then expressed as percentage necrosis. These roots were chopped into small pieces and 5 g sub-samples were mixed with 50 mL of water, and macerated at medium speed for 20 s using a Waring laboratory blender. Nematodes were extracted from the suspension using a Baermann tray for 24 h. The extracts were transferred into 100 mL glass bottles and stored at 4ºC for 24 h to allow the nematodes to sediment. The water volume was standardized to 25 mL by gentle siphoning from the surface. The suspension was homogenized by stirring, and nematodes quantified on a counting dish at ×10 magnifications from triplicate 2.5 mL pipette sub-samples. The nematode densities were expressed as R. similis counts per 100 g of root material.
Statistical analysis Data were analyzed using SAS 9.1 software (SAS Institute Inc. 2001). Diagnostic check for normality was conducted using proc univariate. Since pre-analysis of data from the three replicates yielded similar results in both the phosphorus and potassium experiments, they were all treated as completely randomized. Proc transreg was used to find appropriate Box- Cox transformations, which included generation of suitable powers or lambda (λ) for data (Y) that required transformation. In the phosphorus deficiency experiment, lambda (λ) for root biomass was estimated as 0.75 hence the transformation became Y0.75. Lambda was 1.25 for shoot fresh weight, shoot dry weight, and total biomass. For leaf length and width λ was 2.25. Lambda for number of
134 Effects of endophytic Fusarium oxysporum V5w2 on the root burrowing…...
leaves was 1.75, while for plant height λ was 1.5. Untransformed data were used for total plant biomass, height and R. similis density. In the potassium deficiency experiment, lambda (λ) for root biomass was estimated as 0.5. Lambda was 1.25 for shoot fresh weight, 0.75 for shoot dry weight, 2 for number of leaves, 2.5 for leaf length and 1.5 for leaf width. Untransformed data were used for comparison of total plant biomass, height and R. similis density. Percentage dead roots and percentage necrosis (x) were arcsine√(x/100) transformed in both experiments. Proc glm was used for three-way factorial analyses of variance (ANOVA) among the treatments. Mean separation was conducted using t-tests (LSD) when ANOVA was significant.
Results Phosphorus deficiency experiment (Table 7.1) Most plants exhibited visual symptoms that could be linked to P-starvation, which included dark-green leaves with slightly yellowing margins, while the older leaves curled downwards and dried. Dead roots were mainly found in R. similis-treated plants, and their percentage was higher in sterile than in non-sterile soil. Necrotic lesions were mainly found in plants that were treated with R. similis, and the percentage root necrosis was higher in non- sterile than in sterile soil. Roots of nematode-treated plants that were inoculated with F. oxysporum V5w2 had a higher R. similis density than those that were not inoculated with the endophyte. Roots of nematode-treated plants from sterile soil had a higher density of R. similis than those from non-sterile soil.
135 Chapter 7
Nematode-treated plants in non-sterile soil had greater root biomass than those from sterile soil. Nematode-treated plants were shorter, had lower shoot fresh weight and total biomass than those without nematodes. Plants in non-sterile soil were taller and had more total biomass than those from sterile soil. However, there was no difference in shoot dry weight between treatments. Plants that were grown in non-sterile soil produced more leaves than those from sterile soil. Within sterile soil, nematode-treated plants had fewer leaves than those without nematodes. However, in non-sterile soil, the number of leaves did not vary with nematode treatment. Leaves of plants that were grown in non-sterile soil were longer and wider than those from sterile soil.
Potassium deficiency experiment (Table 7.2) A symptom that could be linked to K-starvation, which was characterized by development of bright-orange coloration on leaf laminae, was evident in the plants. Nematode-treated plants in sterile soil had a higher percentage dead roots and higher R. similis density than those from non-sterile soil. Percentage root necrosis was higher in plants that were inoculated with R. similis than those without the nematode. Root biomass for F. oxysporum V5w2-inoculated plants was lower than for plants that were not inoculated with the endophyte. Root biomass for nematode-treated plants that were grown in sterile soil was lower than for plants from non-sterile soil. Shoot fresh weight, total biomass, plant height, leaf length and width, were lower in plants that were treated with R.
136 Effects of endophytic Fusarium oxysporum V5w2 on the root burrowing…...
similis than in non-treated ones, and higher in non-sterile than in sterile soil. Shoot dry weight was lower in nematode-treated plants than in untreated ones. Nematode-treated plants had fewer leaves than those without nematodes. Plants that were inoculated with F. oxysporum V5w2 and grown in sterile soil had fewer leaves than those that were not inoculated. Plants that were grown in non-sterile soil had more leaves than those from sterile soil.
Discussion In both experiments with the P-starved and K-starved banana plants respectively, exposure to R. similis resulted in a high percentage dead roots and root necrosis. Those were typical effects of damage inflicted by R. similis to banana roots (Viaene et al. 2003; Gowen et al. 2005). Also, in both the phosphorus and potassium experiments, nematode-treated plants were shorter, had lower biomass and produced fewer leaves than those without nematodes. Plants that were treated with R. similis produced smaller leaves compared to those without the nematode under K-deficiency, but leaf size was similar between the two treatments under P-deficiency. Inoculation of the P-starved and K-starved banana plants with F. oxysporum V5w2 did not alleviate plant damage inflicted by R. similis and did not suppress their density in roots at 100 days after exposure to the nematode. Conversely under K-deficiency, plants that were inoculated with F. oxysporum V5w2 had lower root biomass, and produced fewer leaves compared to those without the endophyte. Reductions in root biomass and
137 Chapter 7
e 7.4*** 344 e 36.8 38.5 16.0 16.4 344 e V5w2 and soil sterility on 344 e Height Height Leaves Number Length Width 344 fwt e 191.1 b a 233.4 31.8 b a 33.9 5.0 b a 5.5 Total 343 dwt e Fusarium oxysporum Fusarium 16.1 17.0 Shoot fwt 344 e
Shoot 344 fwt e , the fungal endophyte endophyte fungal the , Biomass Biomass 51.5 156.5 16.1 208.4 32.3 5.3 37.3 16.1 50.7 165.4 17.0 216.2 33.4 5.2 38.0 16.3 gram gram gram gram cm count cm cm -100 g 343 e count 3820 b
R. similis similis R. 5896 a 337 e Radopholus similis Radopholus 341 e percent percent 33.9a b 0.2 38.5a b 0.3 a9510 17.3 b 161 41.8 b a 60.3 18.3 148.9b a 173.1 17.0 19.2 4849 51.1 161.0 16.5 212.3 32.9 5.2 37.7 16.2 density, plant biomass, height, number and size of leaves in tissue culture banana plants supplied with a a with supplied plants banana culture tissue in leaves of size and number height, biomass, plant density, 343 trt 7 1 0.8 1 2.20.7 6.5* 3.1 2.2 3.8 42.1 0.7 0 0.4 44.4 0.7 169.70.9 0.1 0 30.0 0.5 0.4 1.5 43.1 0 2.0 40.6 0.1 1.9 42.0 2.3 40.4 25.6 39.0 35.7 Dead Necrosis Root Dead Necrosis 1 90.3*** 1.5 21.6*** 8.9** 2.9 3.2 3.4 0.1 7.8** 0.8 0.3
1 0 2.3 5.7* 0 2.1 2.6 0.9 1.8 2.6 1.5 0.6 1 1905*** 1599.2*** 111.4*** 81.7*** 15.3*** 2.7 28.4*** 4.4* 33.1*** 3.7 1.3 ) ) R. similis Rs Fo non 16.8 20.1 end nem Effects of the nematode nematode of the Effects
(Fo (Rs S 1 0.2 4.0 3.3 0.5 0.1 0 0.3 0.6 3.1 0 0.5
×
Fo Fo × S × S × × Rs Fo interactions Three-way Rs c.v. (df) Total
Grand mean Soilsterility (S) Two-way 1 interactions Rs 72.0*** 9.9** 19.3*** 37.2*** 1.9 0.5 7.1** 17.6*** 244.9*** 11.7*** 3 Means F values values effects Main Nematode Endophyte F Nematode non Endophyte Source of variation variation of Source df damage Root Table 7.1. Table root damage, damage, root phosphorus deficientnutrient solution
138 Effects of endophytic Fusarium oxysporum V5w2 on the root burrowing…...
35.1 38.6 15.0 37.5 17.0 39.5 15.6 17.2 Asterisks indicate
4.1 c a 5.8 5.0 b a 6.0 dry weight; dry dwt 201.5 b a 223.1 30.8 b a 34.9 4.6b a 5.9 36.3b a 39.1 15.3 b a 17.1 Freshweight and fwt
138.4 159.3 15.7 174.1 16.4 172.2 172.0 17.8 210.0 16.3 29.5 230.6 34.1 236.2 32.1 35.7 156.2 165.7 16.7 16.4 Error degrees offreedom; e 58.6 174.5 17.3 233.1 34.3 5.4 38.8 16.4 62.0 171.7 16.8 233.7 33.5 5.6 38.2 16.4 42.9 156.4 16.7 199.2 32.5 4.9 37.2 16.2 40.8 141.4 15.4 182.9 31.0 5.0 36.5 15.8
Treatment and trt ; 230c 13476 a 5498b c51 32.9 c 50.7 b c277 56.6 ab 64.0 a 93 c 7330b 11768 a
0.05), Means significant 0.05), for effects are boldindicated in font;are not the those with same letter significantly V5w2 ≤ 35.6 41.3 0.1 0.5 0.01, *p *p 0.01, ≤ 44.9 a 44.9 b 23.1 0 c c 0.4 22.3 a 11.7 b 17.6 b 6725a 20.8 a 2918b 44.8 b 57.4 a Fusarium oxysporum oxysporum Fusarium 0.001, **p 0.001,
≤ end 1
0 0.2 34.7 36.8 Non-sterile 11.4 Non-sterile 21.2 12.1 3085 20.3 4862 57.6 57.1 159.1 15.8 172.4 216.7 17.0 33.8 229.5 6.0 36.0 38.4 5.8 16.8 39.8 17.4 non 0.4 0.4 Fo non 33.2 40.1 Fo Radopholus similis; similis; Radopholus Sterile Nematode Non-sterile non Sterile Non-sterile Endophyte Sterile non 23.2 Sterile 15.3 8610 21.5 45.2 19.9 4862 154.0 44.4 16.3 200.0 158.5 30.7 17.1 4.6 202.9 36.3 30.8 15.4 4.6 36.3 15.2
non
significant effect (***psignificant Soil Sterile Non-sterile Nematode nem different (p > 0.05)
139 Chapter 7
e 355 e
42.3 19.3 42.1 19.0
355 e
5.9 a 5.7 b
V5w2 andsoil sterility 355 e Height LeavesHeight Number Length Width fwt 356 e 218.0 b 36.1 b a 246.9 a 40.4 5.5 b a 6.2 40.1b a 44.3 17.9 b a 20.4 Total dwt Fusarium oxysporum 353 e 19.3 19.0 Shoot fwt 350 e 186.8 19.1 238.0 38.1 181.1 19.1 226.5 38.3 Shoot fwt 353 e
51.2 a 45.4 b Biomass Root , thefungal endophyte gram gram gram gram cm count cm cm -100g 355 e 5132a 4097 b 43.5 b 53.2 a 174.6 b 193.6 a R. similis 350 e Radopholus similis Radopholus 12.5 12.0 352 e density, plant biomass, height, number and size of leaves in tissue culture banana plants supplied supplied plants banana culture tissue in leaves of size and number height, biomass, plant density, 11.9 a 11.9 b 6.6 9.4 12.9 6934 18.7a 24.40.2b a 0.6 b 11812a 435 b 40.7 b 55.6 a 168.9 b 198.2 a 18.0 b 20.2 a 209.4 b 36.8 253.8 b a 39.6a 5.7 b 6.0 a 41.1b 43.2a 18.8 b 19.5 a
355 trt 7 percent percent count Dead Necrosis 1 1 0 1 12.5*** 0.1 0.6 0.1 1.9 1.3 16.7*** 5.4* 0.1 2.1 2.1 0.7 106.6 1.1 0 0 105.7 3.7 2.3 1.3 173.6 0.6 2.0 0.2 3.2 17.2 2.3 0.4 0.5 1.5 31.8 0.5 4.1* 23.5 1.4 0.7 0.8 25.9 0.6 19.5 26.3 28.8 18.5
R. similis R. 1 0.4 0.3 2.2 11.2*** 1.0 0 2.7 0.1 7.2** 0.8 0.4
1 305.8*** 252.6*** 102.6*** 77.5*** 35.2*** 12.3*** 47.6*** 12.7*** 7.5** 17.1*** 14.1*** ) ) end nem
non 9.1 11.6 5132 Fo Rs Effects ofthe nematode
(Fo (Rs S × 1 0.5 3.5 0.3 0.3 0.5 0.5 0.2 0.1 3.8 0.5 0.7 Fo Fo × S × × S × Soil Sterile Non-sterile
Endophyte Means F values variation of Source df damage Root values Main effects Nematode Endophyte F Nematode non Grandmean 9.3 12.3 6028 48.3 183.9 19.1 232.2 38.2 5.8 42.2 19.2
Soil sterility (S) interactions Two-way 1 Rs 9.9** Rs 0.1 Fo 11.1** interactions Three-way Rs 32.9*** 12.1*** 0.2 c.v. (df) Total 19.2*** 29.6*** 64.5*** 52.4*** 53.9*** Table 7.2. damage, on root potassiumwith deficient a nutrient solution
140 Effects of endophytic Fusarium oxysporum V5w2 on the root burrowing…...
Asterisks Asterisks
39.8 44.4 17.8 40.4 20.3 44.3 18.1 20.5 dry weight; dry dwt 5.3 d 6.1 a c5.7 6.2 a Fresh weight and fwt
203.8 20.0 262.9 41.2 6.2 45.0 20.7 192.5 20.4 244.7 38.0 5.7 41.4 18.3 182.4 17.8 229.1 39.5 6.1 43.6 20.1 156.6 18.1 191.4 34.2 5.3 38.8 17.5 Error degrees of freedom; e
Treatment andTreatment 0.05), 0.05), Means for significant effects are indicated bold in font; those with the same are letter ≤ trt ;
850c 59.1 a 11c 52.1 ab 7612 b 46.9 b 15734a c 34.8 V5w2 0.01, *p ≤ 1.0 0.3 24.1 24.7
0.001, **p ≤
0.3 c 0.1 c 13.4 b 18.5 24.8 0.4 13228 0.9 a 23.7 497 39.6 168.6 51.3 18.0 193.6 208.2 20.2 37.1 244.8 5.6 39.5 40.7 5.8 43.3 18.6 19.5 Fusarium oxysporum end
Non-sterile 6.2 Non-sterile 12.2 7.0 4861 11.9 3324 51.1 195.1 55.5 19.2 192.1 246.2 18.8 40.7 247.6 40.1 Fo non Fo non 19.0 24.0 0 10329 0.4 377 41.8 169.2 59.9 17.9 202.7 210.7 20.2 36.4 262.6 5.7 39.7 41.5 6.1 43.1 18.9 19.6 Radopholus similis; similis; Radopholus Endophyte Sterile non 12.6 Sterile 13.7 9007 11.2 39.9 11.3 167.3 6861 19.0 47.0 207.2 181.8 35.9 19.5 228.9 36.3 Non-sterile non Sterile Non-sterile Nematode non Nematode Sterile nem indicate significant effect (***p effect significant indicate > 0.05) (p different significantly not
141 Chapter 7 number of leaves in the current work indicate that F. oxysporum V5w2 may have negative effects on banana plant growth when potassium is depleted from soil. Under P-deficiency, the density of R. similis in roots was higher in plants that were co-inoculated with the nematode and F. oxysporum V5w2 compared to those treated with R. similis only. Back et al. (2002) mentioned that nematode penetration may increase in roots that have previously been subjected to fungal enzymes. The question in the present work is why R. similis density was enhanced in plants that were treated with F. oxysporum V5w2 under P-deficiency and not under K-deficiency. In the two experiments, R. similis density and percentage dead roots were lower in nematode-treated plants that were grown in non-sterile than in sterile soil, which may be attributed to antagonistic activities of microbes against the nematode. This may have resulted in better plant growth in terms of height, biomass, number and size of leaves in non-sterile compared to sterile soil. Under K-deficiency, the percentage root necrosis was not different between sterile and non-sterile soil. However, under P-deficiency, the percentage root necrosis was higher in non-sterile than in sterile soil, which seems to contradict the fact that root death was less under these conditions. In these experiments, R. similis damages were lower and plant growth better in non-sterile than in sterile soil. The experiments do not provide evidence for R. similis control by F. oxysporum V5w2 in banana plants. Rather, F. oxysporum V5w2 enhanced nematode colonization in banana roots that were treated with P-deficient nutrient solution; and
142 Effects of endophytic Fusarium oxysporum V5w2 on the root burrowing…...
lowered root biomass and number of leaves in those that received K- deficient solution.
Preparation of media for tissue culture banana plants (Jane and Elvis)
Acknowledgements This work was conducted under the management of the International Institute of Tropical Agriculture (IITA) in Uganda. The funds were provided by the German Ministry of Economic Cooperation and Development (BMZ) and Wageningen University. Technical support that was provided by Jane Luyiga, Phillip Abidrabo, Patrick Emudong, Elvis Mbiru, Fred Kato, Victoria Naluyange, Rose Khainza and Juliana Nakintu. Dr. Phillip Ragama advised on statistical analysis. We give credit to the Soil Chemistry Laboratory of Kawanda Agricultural Research Institute (KARI) for the training and analysis of soil samples.
143
Chapter 8
Effects of endophytic non-pathogenic Fusarium oxysporum V5w2 and mulching on field-grown tissue culture banana plants and root infection by Radopholus similis
A patrol on the banana field experiment (Dennis)
Dennis M.W. Ochieno · Marcel Dicke · Thomas Dubois · Danny Coyne · Piet van Asten · Philip E. Ragama · Arnold van Huis
Chapter 8
Abstract This study assessed the effects of endophytic non-pathogenic Fusarium oxysporum V5w2 and mulch on Radopholus similis and other nematodes on banana plants in the field. The experiment was a 3-factor (2 × 2 × 2) complete randomized block design with or without the endophyte, R. similis or mulch. Root damage was higher when plants were treated with R. similis compared to non-treated plants, while plant height, leaf size and bunch size were smaller in R. similis-treated plants. Plant growth and bunch size were greater in mulched than in unmulched plots. Endophyte-treated plants were short, with few leaves that were small, and had shorter duration to harvest than endophyte-free ones. When R. similis-treated mulched plants were inoculated with F. oxysporum V5w2, plant toppling occurred less frequently compared to plants that were not treated with the endophyte, probably because the plants were smaller during growth. In conclusion, the benefits of mulching in banana production were evident, while the enhancing effect of inoculating tissue culture plants with F. oxysporum V5w2 could not be verified.
146 Effects of endophytic non-pathogenic Fusarium oxysporum V5w2 and……
Introduction The production of highland cooking banana (Musa spp. AAA) in Africa has been constrained by a complex of plant-parasitic nematodes especially Radopholus similis, Helicotylenchus spp., Pratylenchus spp. and Meloidogyne spp. (Barekye et al. 2000; Talwana et al. 2000). Control of these pests has been difficult as banana plants are mainly propagated through suckers that are usually infested. Apart from being less productive, nematode-infested suckers are the main mode of widespread dissemination of banana pests (Marin et al. 1998; Price 2006). There have been efforts to manage nematodes through pest-free planting material and mulching of banana fields. Banana plantations, especially those established from pest-free planting material, produce high biomass and yield when mulched (McIntyre et al. 2000, 2003). Tissue culture technology has become important for rapid multiplication of pest- free banana planting material (Krikorian and Cronauer 1984; Vuylsteke 1998). However, infestation of tissue culture plants by pests in the field remains a problem. Tissue culture techniques eliminate pest-suppressive endophytic microbes, which contributes to the vulnerability of banana plants to infection (Sikora et al. 2000). Therefore, research is being conducted to introduce endophytic microbes, such as non-pathogenic Fusarium oxysporum, into tissue culture banana plants for protection against R. similis and other pests (Dubois et al. 2006a; Pocasangre 2006; Sikora et al. 2008). Fusarium oxysporum strain V5w2 has been shown to be suppressive against R. similis in banana plants under screenhouse conditions (Athman et al.
147 Chapter 8
2007). Several strains of non-pathogenic F. oxysporum have been found to suppress R. similis under field conditions (zum Felde 2008, zum Felde et al. 2009; JKUAT 2009). Mulching is one of the cultural methods used in banana culture. However, studies to address the interaction of mulching and endophyte treatement have never been carried out. The objective of the current study was to investigate the combined effect of F. oxysporum V5w2 and mulch against R. similis and other nematodes in tissue culture banana plants under field conditions.
Materials and Methods Experimental design The field experiment was conducted between October 2006 and February 2009 at the International Institute of Tropical Agriculture (IITA), Namulonge, Uganda. The location has isohyperthermic Rhodic Kandiudalf soils, lies 0.53 N, 32.58 E, 1150 m above sea level, with an average annual rainfall of 1200 mm, the first rainy season lasting from March to June and the second rainy season between September and January (McIntyre et al. 2000). The experiment was a three factor (2 × 2 × 2) randomized complete block design of banana plants with and without F. oxysporum V5w2 inoculation, with and without R. similis treatment, and grown on mulched and unmulched plots. The eight treatment combinations were laid out in eight plots (15 × 12 m), separated by 3 m slashed land in a block (144 × 12 m). The experimental site was located on a gentle slope. The three blocks (replicates), each running across the slope, were consecutively separated along the slope by strips (10 m wide) of slashed land. Blocking across the
148 Effects of endophytic non-pathogenic Fusarium oxysporum V5w2 and……
slope was meant to cater for possible variations due to land inclination. The field was prepared during the dry season in December 2006. Each plot had 20 planting holes (40 cm diameter, 40 cm depth), which were spaced at 3 × 3 m giving a total of 480 planting holes in the field. Border rows of banana plants were grown surrounding the three blocks at distances of 3 m from the peripheral planting holes. Cow manure (10L) was applied in every hole before planting.
Inoculation of banana plants with Fusarium oxysporum V5w2 and Radopholus similis Tissue culture banana plants (genomic group AAA-EA, cv. Kisansa) that had been maintained in rooting media for 1 month were obtained from Agro-Genetic Technologies (Buloba, Uganda). The 480 plants were maintained under hydroponic conditions in 250 mL cups containing a nutrient solution (1 g/L, PolyFeed, Haifa Chemicals, Israel) in a humidity chamber for 8 weeks. The plants were graded into four sizes and distributed over 24 groups of 20 plants each. Twelve groups were marked for inoculation with the endophyte and the other 12 groups were unmarked. A loamy forest soil had been collected from central Uganda. The soil was sieved (5 mm aperture) before being thoroughly mixed. The soil was sterilized at 100ºC for 1 h using an electrode steam conditioner (Marshall- Fowler, South Africa), and packed in 2 L plastic bags. Wild-type F. oxysporum V5w2 endophyte was obtained as a soil culture from IITA in Namulonge. This endophyte was part of a collection that was obtained from surface-sterilized banana roots in Uganda by
149 Chapter 8
Schuster et al. (1995). The endophyte inoculum was formulated as a solid substrate in maize bran as described by Paparu et al. (2006b). The banana plants were inoculated and grown in the plastic bags after sprinkling 2 g of the F. oxysporum V5w2 inoculum onto roots and into the planting holes. The controls were treated with 2 g of sterile maize bran that did not contain the endophyte. The plants were watered daily in the screenhouse. Radopholus similis were originally obtained from banana fields at Namulonge and maintained aseptically on carrot discs (Speijer and De Waele 1997). The nematodes were supplied in sterile water suspension containing mixed stages of 250 R. similis / mL. Fifty days after planting in plastic bags, three holes (5 cm deep) were made into the soil around the base of each plant, by using a disinfected stick. Each plant marked for nematode treatment was inoculated with ~500 R. similis in 2 mL of the suspension distributed across the three holes before covering them with soil.
Planting and sampling in the field The 18th of January 2007, thirty days after inoculation with R. similis, the banana plants were planted in the field. The plants were drenched with 5 L of water thrice every week since it was the dry season in Central Uganda. Dry grass mulch was applied within a week to the respective treatments to a thickness of approximately 10 cm. The mulch and manure (10 L per plant) were reapplied after 6 months. Mulched plots were maintained by hand- weeding while unmulched ones were weeded using a hoe. Plants were desuckered to ensure that only one sucker remained from each crop cycle. Data were collected for the mother plants only i.e. the ones that received
150 Effects of endophytic non-pathogenic Fusarium oxysporum V5w2 and……
treatments but not their suckers. Plant growth parameters, which included height, number and size of leaves, were recorded at planting and after each of the next four months (i.e. 41, 69, 97 and 125 days). At 175 days after planting, roots were sampled from five randomly selected plants per plot for quantifications of nematode densities and root damages according to Speijer and De Waele (1997). Root damage assessment included percentage dead roots, percentage root necrosis, ratings for feeder root damage, and the occurrence of root knots. Four roots, each of which was sectioned into six pieces, were also randomly selected from five plants per plot (i.e. 15 plants per treatment) to verify whether inoculation with wild-type F. oxysporum V5w2 contributed to root colonization by Fusarium spp. in general. We used the percentage root piece colonization method of Paparu et al. (2006a), except that our PDA media was non-selective. Plants that toppled before harvest were recorded and expressed as percentage of the total plants, after excluding those that were lost to other factors (not linked to R. similis damage). Bunches were judged mature by an experienced field assistant, based on loss of their green coloration, and finger filling that makes banana fruits to appear cylindrical as the corners on their surfaces become blunt. The height of a plant bearing a mature bunch, the girth i.e. circumference of the pseudostem at 100 cm from the ground, and the number of functional leaves were recorded. After recording the harvest date, a plant was cut down and the diameter of its base was recorded. The bunch weight, number of hands and their fingers were recorded. Plants whose bunches were harvested out of the initial plant population per treatment were recorded.
151 Chapter 8
Data analysis Data were analyzed using SAS 9.1 software (SAS Institute Inc. 2001). Diagnostic check for normality was conducted using proc univariate. Proc transreg was used to find appropriate Box-Cox transformations, which included generation of suitable powers or lambda (λ) for data (Y) that required transformation. For pre-harvest data, λ for leaf length was estimated as -0.25 hence the transformation became Y-0.25 while for width λ was 0.25. Plant heights were log transformed. Percentage dead roots and necrosis (x) were arcsine√(x/100) transformed. Data for number of leaves and nematode densities were not transformed. The means for the five stages of sampling for data on plant height, number and size of leaves were considered for analysis. For harvest data, λ for height and bunch weight was 0.75, for girth λ was 0.5, and -0.25 for diameter. Percentage dead roots and necrosis were analyzed by three-way analyses of variance (ANOVA) using proc glm. Proc mixed was used for three-way ANOVA for pre-harvest plant height, number and size of leaves, and nematode densities; and at harvest for height, bunch weight, girth and diameter. Mean separation was done by t- tests (LSD) when there were significant differences between treatments. Number of leaves at harvest, time until harvest, banana hands and fingers and pre-harvest data on feeder root damage were analyzed by three- way ANOVA on ranks in proc glm and the means separated by Lsmeans. Occurrence of root knots, toppled and harvested plants, and percentage root piece colonization by Fusarium spp. were analyzed by proc genmod as data having a binomial distribution. Bonferroni-adjusted P-values were obtained by proc multtest and used for pairwise comparisons on toppled and
152 Effects of endophytic non-pathogenic Fusarium oxysporum V5w2 and……
harvested plants. Proc corr was used for correlations between the assessed parameters. There were significant effects of blocks (Tables 8.1 and 8.2), which confirms the need to replicate the experiment across the slope.
Results Pre-harvest observations (Table 8.1) General observations on plants in the screenhouse revealed that plants were characterized by slight yellowing of leaves that appeared to have mild wilting. These symptoms, which occurred especially in the endophyte- treated plants, were not quantified. The plants appeared to have recovered by the time of planting in the field. After 125 days in the field, plant height, and size of leaves were lower in plants that were treated with R. similis or F. oxysporum V5w2 compared with those without either of the two organisms. The number of leaves of plants treated with the endophyte was also lower than that of plants not treated with the endophyte. Mulched plants were taller and produced more leaves that were larger in size compared to unmulched plants. After 175 days in the field, densities of R. similis were very high in plants that were treated with the nematode while they were almost absent in the untreated ones. Other nematode groups were also recorded in the root samples and included Helicotylenchus spp. and Meloidogyne spp. but no treatment effects were observed. After 175 days, the percentage dead roots and necrosis were higher in plants that were treated with R. similis compared to those without the nematode. Feeder root damage was higher in mulched plots than in plots
153 Chapter 8 without mulch for plants that were not inoculated with R. similis, but was not different in those treated with the nematode. Root knots occurred in 78 % of the plants (n = 116) but did not vary between treatments (Genmod, χ2 test, p > 0.05). Fusarium spp. were recorded in all treatments with percentage root piece colonization varying significantly, but the trends could not be linked to any of the treatments.
Observations at harvest (Table 8.2, Figure 8.1) Plants inoculated with F. oxysporum V5w2 took shorter time from planting to harvest than those not treated with the endophyte. Mulched plants had more leaves, were taller with wider girth and corm diameter than the unmulched ones. Plants treated with R. similis had narrower girths and had bunch sizes with less hands than those without the nematode. Mulched plots produced heavier banana bunches with more hands and fingers than unmulched ones. Toppling was significantly higher in R. similis-treated plants that were mulched compared to other groups, except for those treated with both the nematode and F. oxysporum V5w2 on mulched plots that were intermediate in toppling and not different from other treatments (χ2 test, p < 0.0001) (Fig. 8.1). Lowest number of bunches were harvested from R. similis-treated plants that were mulched compared to other groups; those treated with both the nematode and F. oxysporum V5w2 on mulched plots were intermediate in bunch production and not different from other treatments (χ2 test, p < 0.0001) (Fig. 8.1). Among the assessed parameters (time until harvest, plant height, girth, diameter, number of leaves, bunch
154 Effects of endophytic non-pathogenic Fusarium oxysporum V5w2 and……
weight, number of hands and fingers), most were positively correlated (p < 0.05) except for the absence of correlation between time until harvest with bunch weight, diameter and number of leaves (p > 0.05).
Table 8.1. Effects of endophytic Fusarium oxysporum V5w2, the nematode Radopholus similis and mulch on plant height, number and size of leaves, nematode densities and root damage in
tissue culture banana plants grown under field conditions
Source of variation 5 125 days after planting 175 days after planting df num Height Leaves Nematode densities Root damage Number Length Width R. similis Helicoh Melom Dead Necrosis FR fr
F values Main effects Block 2 2.4 4.7* 1.19 2.54 0.32 3.51 1.18 0.49 2.91 0.03 Nematode (Rs) 1 4.92* 1.94 7.27* 6.14* 9.44** 0.82 1.15 9.01** 65.1*** 0.47 Endophyte (Fo) 1 5.02* 8.1* 9.29** 9.74** 0.83 3.92 1.08 0.11 3.85 0.66 Mulch (M) 1 13.6* 15.3** 13.76** 14.47** 1.18 2.47 1.47 2.18 0.06 2.17
Two-way interactions Rs × Fo 1 0.9 0.46 0.64 1.0 0.87 0.09 1.57 0 4.39 0.57 Rs × M 1 0.31 0.27 0.22 0.41 1.43 4.14 1.29 0.3 0.63 6.76* Fo × M 1 1.86 0.04 0.28 0.4 0.45 0.26 1.63 0 1.18 0
Three-way interactions Rs × Fo × M 1 0.18 0.41 0.16 0.31 0.45 1.34 1.1 0.16 0.34 1.34 df den 14 14 14 14 14 14 14 14 14 14
Means cm count cm Count -100g percent rank Grand mean 53.1 180 6.2 57.4 28.1 715 75 918 2.9 7.2 2.3
Nematode Rs 50.2 b 6.1 54.5 b 26.9 b 1333 a 93 340 4.7 a 14.0 a 2.4 non 56.0 a 6.3 60.2 a 29.4 a 33 b 58 1558 1.3 b 0.9 b 2.3
Endophyte Fo 50.7 b 5.9 b 54.6 b 26.8 b 863 118 1418 2.9 9.2 2.3 non 55.6 a 6.4 a 60.2 a 29.5 a 558 30 383 2.9 5.4 2.4
Mulch mulched 58.4 a 6.5 a 61.9 a 30.2 a 440 40 1583 2.0 6.1 2.4 not 47.8 b 5.9 b 52.9 b 26.1 b 983 110 278 3.9 8.3 2.3
Nematode Fo 46.7 5.7 50.9 25.1 1670 143 230 4.6 18.5 2.3 non 53.8 6.3 58.1 28.8 998 43 450 4.7 10.0 2.5 non Fo 54.6 6.1 58.2 28.5 28 93 2645 1.5 10.1 2.3 non 57.4 6.4 62.2 30.2 35 15 305 1.2 0.8 2.3
Nematode mulched 55.7 6.3 59.1 29.1 800 15 385 3.7 11.3 2.3 ab not 44.8 5.8 50.0 24.8 1833 168 298 5.7 16.6 2.5 a non mulched 61.2 6.6 64.7 31.2 53 68 2863 0.4 1.3 2.5 a not 50.9 5.9 55.7 27.5 10 45 253 2.3 0.6 2.1 b num Numerator degrees of freedom; den Denominator degrees of freedom. h Helicotylenchus spp. m Meloidogyne spp. 180 Maximum height fr Feeder root damage; Asterisks indicate significant effect (***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05); Means for significant effects in bold font; those with the same letter are not significantly different (p > 0.05)
155 Chapter 8
Table 8.2. Effects of endophytic Fusarium oxysporum V5w2, the nematode Radopholus similis
and mulch on time until harvest (days), plant growth, number of leaves and bunch size in tissue
culture banana plants grown under field conditions Source of variation df num Time t Plant growth Leaves Bunch size Height Girth Diameter Hands Fingers Weight
F values Main effects Block 2 0.28 2.17 4.6* 3.94* 8.45** 2.05 4.43* 5.55* Nematode (Rs) 1 0.70 2.73 7.2* 4.27 0.76 5.57* 3.09 2.05 Endophyte (Fo) 1 6.60* 0.09 1.9 1.07 0.93 0.87 2.40 0.79 Mulch (M) 1 1.88 38.0*** 65.2*** 30.7*** 20.71*** 25.32*** 41.07*** 42.7***
Two-way interactions Rs × Fo 1 2.01 1.31 1.74 0.42 0.14 2.86 1.54 0.69 Rs × M 1 2.21 0 0.04 2.51 4.09 0.03 0.27 0.16 Fo × M 1 0.23 2.96 1.14 0.05 0 1.55 1.81 1.6
Three-way interactions Rs × Fo × M 1 0.06 0.04 0.12 0.01 0.76 0.24 0.68 0
df den 14 14 14 14 14 14 14 14
Means
days cm count kg Grand mean 517 258 46.1 20.6 3.7 6.1 87.8 10.1
Nematode Rs 522 239 42.6 b 19.3 3.7 5.6 b 75.4 8.8 non 514 271 48.6 a 21.5 3.7 6.5 a 96.5 11.0
Endophyte Fo 490 b 259 45.9 20.5 3.6 6.1 87.7 10.3 non 546 a 257 46.4 20.7 3.7 6.2 87.9 9.9
Mulch mulched 519 284 a 51.5 a 22.5 a 4.2 a 6.8 a 102.4 a 12.4 a not 514 230 b 40.5 b 18.6 b 3.1 b 5.5 b 72.5 b 7.7 b num Numerator degrees of freedom; den Denominator degrees of freedom. t Time from planting until harvest; Asterisks indicate significant effect (***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05); Values for significant effects in bold font; those with the same letter are not significantly different (p > 0.05)
156 Effects of endophytic non-pathogenic Fusarium oxysporum V5w2 and……
a 100 60
90 ab 60
80 ab ab b 60 60 60 70 b x 60 60 b 58 60
50 c 60 40 xy 56 30
20 y Percentage of banana plants toppled or harvested y 60 y 10 49 y 54 y 34 y 42 58 0 O N E M EN NM EM ENM
Toppled plants Harvested plants
Figure 8.1. Percentages of banana plants that toppled prematurely and those whose bunches were harvested in a three factor (2 × 2 × 2) randomized complete block design field experiment. The treatments included the control without any treatment (O), the nematode Radopholus similis (N), the endophyte Fusarium oxysporum V5w2 (E), mulch (M) and their respective combinations EN, NM, EM and ENM. Numbers above the bars represent sample sizes. Bars with the same letter for toppled plants (x,y) and for harvest (a,b,c) are not significantly different (Genmod, χ2 test, Bonferroni adjusted, p < 0.05).
157 Chapter 8
Discussion In the present study, R. similis expressed its pest potential in banana plants. This was evident from high root injury that resulted in poor plant growth and fewer banana hands. These are typical symptoms of R. similis infection (Speijer and De Waele 1997). Banana plants that were grown on mulched plots were larger with heavier bunches than those without mulch. This enhanced plant growth on mulched plots has been associated with improved soil fertility and water uptake by the banana plants (McIntyre et al. 2000). However, the number of bunches produced by R. similis-treated plants from mulched plots was lower than from other treatments due to higher rate of toppling. This was an indication of weakened anchorage of the large plants by R. similis-damaged roots (Gowen et al. 2005), and was evident in high injury to feeder roots in mulched plots. Apart from R. similis, other root parasitic nematodes that were recorded included Meloidogyne spp. and Helicotylenchus spp. There was no evidence on endophytic control of the nematodes. Fusarium oxysporum V5w2-inoculated banana plants exhibited reduced performance as was apparent from poor growth in the form of being short, having few leaves of small size, and a shorter time to harvest. Plant growth suppression by endophytes has been mentioned by Paparu et al. (2009b). Inoculation treatment with F. oxysporum V5w2 seemed to reduce toppling in R. similis-treated plants on mulched plots, resulting in the production of more bunches compared to those without the endophyte. This could be because the F. oxysporum V5w2-treated plants on mulched plots were smaller during growth, making their R. similis-damaged roots sustain
158 Effects of endophytic non-pathogenic Fusarium oxysporum V5w2 and……
lesser strain than those without the endophyte. The endophyte-treated plants may also have been harvested earlier before toppling since they took shorter time to harvest than those without the fungus. Growth suppression on young potted banana plants did not occur in other studies that utilized F. oxysporum V5w2 (Niere et al. 1999), even under dual inoculation with other strains (Paparu et al. 2009b). The question in the current study is why growth suppression by F. oxysporum V5w2 occurred in mature banana plants in the field. Probably, the unusual capacity of F. oxysporum V5w2 as a non-pathogenic endophyte, to invade the xylem through root injuries (Paparu et al. 2009a), may be a clue on its possible relationship with undesirable strains that invade the xylem via deep wounds (Raut and Ranade 2004). Young banana plants infected with such undesirable strains rarely express the symptoms (Ploetz 1998). However, a considerable number of the plants in the current study became weak after inoculation while still young. There may also be indirect effects of non-pathogenic F. oxysporum that result in reduced performance of banana plants. For instance, non- pathogenic F. oxysporum strain BRIP 45952 increased the activities of pathogens in banana plants (Forsyth et al. 2006). Such interactions cannot be ruled out in the current study without proper identification of F. oxysporum strains. However, we could not discern the wild-type F. oxysporum V5w2 from Fusarium spp. that occurred in root samples from all treatments and the control. This is because the method we used does not guarantee identification of the wild-type F. oxysporum V5w2 (Paparu et al.
159 Chapter 8
2009a), among numerous Fusarium spp. that exist in banana fields (Nel et al. 2006). In this study, R. similis inflicted damage on banana roots, constrained plant growth and caused a reduction in number of banana hands on bunches. Mulch promoted banana plant growth and bunch size, but increased toppling of R. similis-treated plants. The F. oxysporum V5w2- treated plants expressed signs of constrained growth that could have resulted in less toppling, and shortened duration to harvest. In conclusion, the benefits of mulching in banana production were evident, while the enhancing effect of inoculating tissue culture plants with F. oxysporum V5w2 could not be verified.
Field maintenance (George, Katerigga, Ziggy, Lubowa and Kato) Acknowledgements This work was conducted under the management of the International Institute of Tropical Agriculture (IITA) in Uganda. The funds were provided by the German Ministry of Economic Cooperation and Development (BMZ) and Wageningen University and Research Centre (WUR).
160 Chapter 9
Summarizing discussion
Discussions during a field excursion (Dennis and Arnold)
Dennis M.W. Ochieno Summarizing discussion
Introduction The banana weevil Cosmopolites sordidus, and the root burrowing nematode Radopholus similis, inflict damage on banana plants. These organisms are among the most serious pests that affect banana production worldwide. There have been efforts to manage the pests through clean planting material produced by tissue culture (Vuylsteke 1998; Gold et al. 2001). Growth and yields of clean banana planting material can be enhanced by mulching (McIntyre et al. 2000, 2003), and through fertilizer application (Smithson et al. 2001). However, banana plants produced by tissue culture are vulnerable to pest infestation partly because they lack protective endophytic microbes (Sikora et al. 2000; Pocasangre 2006). This has prompted research on the introduction of protective endophytic microbes into tissue culture banana plants (Dubois et al. 2006a). Research on endophyte-enhanced banana tissue culture technology is being conducted by various research groups (Pocasangre et al. 2000; Dubois et al. 2006a,b; Sikora et al. 2008; JKUAT 2009). Non-pathogenic Fusarium oxysporum strain V5w2 has been shown to suppress pests in banana plants, which include R. similis and C. sordidus (Pocasangre et al. 2000; Vu 2005; Vu et al. 2006; Waithira 2009; Paparu et al. 2009b). The studies presented in this thesis have addressed the effects of nutrients and soil microorganisms, as well as mulching, on F. oxysporum V5w2-enhanced tissue culture banana plants. The aim of this chapter is to integrate the results of Chapters 3-8 of this thesis, and to place them in the context of other studies. Knowledge gaps are identified and recommendations for future studies are made.
162 Chapter 9
Overview of the research Knowledge on endophytic F. oxysporum mechanisms of action is still underdeveloped as the field is still new, especially for strain V5w2 (Sikora et al. 2008). Research on F. oxysporum endophytes has recently moved from the laboratory and screenhouse experiments to field trials (Dubois et al. 2006a,b; Vu 2005; Athman et al. 2006, 2007; Paparu et al. 2008, 2009a,b; zum Felde 2008; Sikora et al. 2008; Waithira 2009). There have also been some ongoing farmer-based trials for F. oxysporum V5w2 and other endophytes in Africa, and with some large-scale banana producers in countries like Costa Rica (Dubois et al. 2006b; Pocasangre 2006; Pocasangre et al. 2006). The work reported in this thesis investigated the effects of mulching, nutrients and soil microorganisms on the plant- endophyte-pest interactions. The research is based on basic principles that included mechanisms of action and diagnostic techniques (Fig. 1.3), which had already been developed and approved by predecessor researchers, and considered ready for farmers (see Dubois et al. 2006a,b). Therefore, the research in this thesis addressed the effects of other agronomic practices on the effectiveness of endophytic fungi for biological control of pests in banana.
163 Summarizing discussion
Effects of mulching, nutrients and soil microorganisms on banana plants, Cosmopolites sordidus and Radopholus similis In this thesis, mulched tissue culture banana plants were larger with better bunches than unmulched ones (Chapter 8). Plant growth promotion by mulch has been linked to enhancement of soil moisture and nutrients (McIntyre et al. 2000). Poor nutrition, such as lack of N, P, K, or the deprivation of all essential nutrients constrained the growth of potted banana plants (Chapter 4). Proper nutrition strengthened plant health and made the plants less suitable to pests. Low suitability of banana plants to pests due to proper nutrition was evident from lower R. similis densities in fertilizer- treated plants than in nutrient-deficient ones (Chapter 4). This may provide an explanation for the lower weights of C. sordidus larvae reared on fertilizer-treated plants than on nutrient-deficient plants (Chapter 3). Alternatively, enhancement of soil nutrient content by mulch may have alleviated competition for resources between banana roots and microorganisms. This was apparent in the screenhouse experiments: plant growth was poorer in non-sterile than in sterile soil in plants that were treated with N-deficient solution or water only (Chapters 4 and 6), but was better when N-rich solutions were applied (Chapters 4, 5 and 7). These results suggest that microbial competition for N constrains plant growth (see Kaye and Hart 1997 and Hodge et al. 2000). Also, mulch may have enhanced the activities of pest-suppressive soil microorganisms (Rosemeyer 2001). The suppressive effects of soil microorganisms on R. similis were demonstrated in having lower nematode density and root damage in non-
164 Chapter 9 sterile than in sterile soil (Chapters 4-7). However, we lack an explanation for the enhanced damage to feeder roots in mulched plants (Chapter 8).
Effects of Fusarium oxysporum V5w2 on banana plants, Cosmopolites sordidus, Radopholus similis and other nematodes Principles in the endophyte-enhanced tissue culture banana technology have been developed and approved for farmer’s fields (Fig. 1.3; Dubois et al. 2006a,b). For instance, the biological control potential of F. oxysporum V5w2 and its mechanisms of action have been studied, methods have been developed for establishing in planta colonization by the fungus following inoculation (Dubois et al. 2006a,b; Sikora et al. 2008; Paparu et al. 2009a,b, 2010). In this thesis, I have taken this information as a basis for studying the effects of nutrients, soil microorganisms and mulch, on the control of banana pests by endophytic non-pathogenic F. oxysporum V5w2.
Root inoculation and biological control potential of Fusarium oxysporum V5w2 The damaging effects of C. sordidus, R. similis and other nematodes in tissue culture banana plants are evident in this thesis, as well as in previous studies (Paparu et al. 2009b; Mendoza and Sikora 2009). Therefore, introducing beneficial endophytic microbes, by root-dipping (Chapters 5-7, Paparu et al. 2006b) or using solid substrate inoculum (Chapter 8, Paparu et al. 2006b), was expected to restore plant protection against pests (Dubois et al. 2006a,b; Pocasangre 2006, Pocasangre et al. 2006, 2007). This was only partly confirmed in Chapter 3, in which potted plants that were inoculated
165 Summarizing discussion
with F. oxysporum V5w2 in sterilized soil were less preferred by C. sordidus in the field and had lower damage by weevil larvae in the screenhouse than those without the endophyte. Also, in olfactometer choice experiments, the banana weevil preferred the volatiles from control plants over volatiles from plants that had been inoculated with B. bassiana G41 (Chapter 3). However, the weevils did not discriminate between control plants and F. oxysporum V5w2-inoculated plants in the olfactometer (Chapter 3). Furthermore, F. oxysporum V5w2 was suppressive to R. similis density, but only in N-starved potted banana plants (Chapter 6). These observations confirm some pest-inhibitive effects of F. oxysporum V5w2 and B. bassiana G41 in banana plants that are inoculated with the endophytes under sterile soil conditions. Although the suppression of R. similis by F. oxysporum V5w2 is desirable (Chapter 6), the N-deficient conditions under which this occurred were not favourable as the potted plants were stunted (Chapters 4 and 6). However, the work in Chapter 3 was conducted with sterilized soil only, and lacks supportive data on changes in soil physical and chemical properties, which may have affected the results (Troelstra et al. 2001). The inhibitive effects of F. oxysporum V5w2 and B. bassiana G41 on C. sordidus need to be investigated using plants grown in non-sterile soil to address the effects of soil microorganisms. Establishing whether F. oxysporum V5w2 existed in planta is necessary for associating its inhibitive effects with successful establishment of the endophyte (Paparu et al. 2009a,b).
166 Chapter 9
Establishment of root colonization by F. oxysporum V5w2 In this thesis, four types of experiments were used to address the presence or activity of KClO3-resistant F. oxysporum V5w2 in potted banana plants inoculated with the endophyte. These tests included: (A) Estimation of percentage root piece colonization in selective media, partly done according to Paparu et al. (2006a, 2009a,b,c), in all the experiments within Chapters 5-8 (e.g. see Figure 9.1). However, data from those tests are not reliable as they present a combination of Fusarium oxysporum and other Fusarium spp. This is because finer morphological details of the growing fungi, such as conidial structures and the branching of mycelia, which are used for differentiating F. oxysporum (Leslie et al. 2006), were microscopically observed in some samples and not in others, but with little emphasis due to the large sample sizes. This was further complicated by the fact that the scores (counts) for Fusarium growth were simultaneously recorded by two observers, one of them seemed more reliable in identifying F. oxysporum, while the other one could only identify Fusarium spp. in general based on mycelial growth in PDA (see Figure 5.1). Since data scoring was simultaneous by the two observers on randomly selected pieces of roots, the numerical data for samples with F. oxysporum could not be separated from other Fusarium spp. The graphs therefore give an indication that inoculation with F. oxysporum V5w2 contributed to the overall levels of colonization, but the percentages are inflated by other Fusarium spp. Based on studies conducted by Paparu et al. (2009a,b) on the same location with similar plant cultivars, most of these are presumably
167 Summarizing discussion
a 30 1002 30
a 25 504 25 oxysporum oxysporum
b 1134 20 b ab b b 20 Fusarium Fusarium b 504 Fusarium oxysporum Fusarium 1188 1008 1026 b Fusariumoxysporum 1026 990 15 c 15 c 486 c 504 1026 10 10 d d 504 d 504 468 d 5 5 504 Percentage root piece colonization by by colonization piece root Percentage Percentage root piece colonization by by colonization piece root Percentage Percentageroot piece colonization by Percentageroot piece colonization by 0 0 control nematode endophyte nema+endo control nematode endophyte nema+endo
sterile soil non-sterile soil sterile soil non-sterile soil
Figure 9.1. Percentage root piece colonization by endophytic Fusarium oxysporum (i.e.
combinations of Fusarium oxysporum and other Fusarium species) in tissue culture banana plants
treated with the nematode Radopholus similis only, the endophyte KClO3-resistant Fusarium oxysporum V5w2 only, or combined inoculation with the nematode and the endophyte (nema+endo), and grown in sterile or non-sterile soil. Plants were either treated with N-deficient solution in experiment 1 (Left) or supplied with water only in experiment 2 (Right). Data were
collected from surface-sterilized root pieces that had been plated on KClO3-containing media according to Paparu et al. (2006a). Data were scored by two observers, the first one basing on the growth of whitish mycelia with pink pigmentation and also seemed to score for F. oxysporum based on mycelial and conidial structure using the Fusarium Laboratory Manual (Leslie et al. 2006); the second observer only basing on whitish mycelia with pink pigmentation (see the mycelia growing from root pieces in Fig. 5.1). Bars with the same letter not significantly different (genmod, χ2 test, p > 0.05; sample sizes indicated above bars). Therefore, the graphs give inflated values for Fusarium oxysporum due to contaminants, while the contribution of V5w2 inoculation to the levels of colonization can be considered minimal especially when the nematodes were present.
168 Chapter 9 contaminations that comprise 5-10%. Hence, the endophyte did not establish properly, except in the nematode treatment, where it seemed to have established, probably due to nematode inflicted wounds (Back et al. 2002). However, although similar tests were conducted for field plants (Chapter 8), the data were heavily masked by contaminants; furthermore the endophyte used was wild-type F. oxysporum V5w2 that could not be identified by the method (Paparu et al. 2009a). Therefore, in Chapter 8, I scored for all Fusarium spp. based on mycelial growth on media, to check whether the addition of F. oxysporum V5w2 contributed to the overall level of colonization, this data did not indicate the establishment of the endophyte. (B) The data on optical density of root extracts in Chapter 5 gave only circumstantial evidence on the activity of the endophyte in inoculated roots (see Fig. 5.2). (C) Some of the contaminant Fusarium and other fungi from ‘A’ above were tested in vitro and some were found to inhibit the endophyte (see Chapter 5). Therefore, they may have contributed to the outcomes, probably by inhibiting the establishment of the endophyte. There were some fungi that did not interact inhibitively with F. oxysporum V5w2, which may have comprised the endophyte that was inoculated into the roots. In this thesis, the application of F. oxysporum V5w2 as a single inoculum in banana plants has apparently been unsuccessful in suppressing R. similis. However, other authors have mentioned successful control of R. similis in banana roots using F. oxysporum V5w2 as a single inoculum (Vu et al. 2006; Athman et al. 2007; Waithira 2009). Suppression of both C. sordidus and R. similis has also been achieved through dual inoculation with
169 Summarizing discussion
F. oxysporum strains V5w2 and Emb2.4o in potted banana plants grown in sterilized soil without nutrient stress (Paparu et al. 2009b). The absence of full support for the control of nematodes by F. oxysporum V5w2 is likely to have been caused by ineffective inoculation of banana roots with the endophyte. I had assumed that inoculation was successful because I had employed the inoculation method developed by Paparu et al. (2006a, 2009a). However, in contrast to Paparu et al. (2006a, 2009a), I did not damage the roots before inoculation and this may have interfered with successful inoculation. Some other aspects of the experimental setup have been changed as well, such as inoculation of relatively young plants and the use of relatively small pots in Chapters 5-7; their effects on endophyte colonization remain to be elucidated.
Plant growth suppression The application of F. oxysporum V5w2 did not promote plant growth and was not suppressive to R. similis when CNS, water only, P- or K-deficient solutions were applied, and in the field (Chapters 5-7, Chapter 8). Conversely, in the field, plants that were inoculated with F. oxysporum V5w2 had poorer growth in the form of being shorter with fewer and smaller leaves compared to those without the endophyte (Chapter 8). The plants also took shorter time to harvest (Chapter 8), which may be an indication of premature ripening. These effects probably occurred because the young plants used in Chapter 8 were in a poor condition after inoculation, while some roots may have died, hence interfering with in planta establishment of the endophyte, and potentially compromising the
170 Chapter 9 plants instead of enhancing them. Fusarium oxysporum V5w2-treated plants grown under K-deficiency had poor growth under screenhouse conditions (Chapter 7). The low K levels in soils at the experimental site (Chapters 4- 7), and generally in Eastern Africa (van Asten et al. 2004), may be a contributing factor to the negative effects of F. oxysporum V5w2 on banana plants. Growth suppression in endophyte-treated plants has been mentioned by Paparu et al. (2009b). East African highland cooking banana cultivars like those used in this thesis exhibit resistance to F. oxysporum (Kangire et al. 2000), while young banana plants rarely express symptoms of infection with such endophytes (Ploetz 1998; Raut and Ranade 2004). These may explain why plant growth suppression by F. oxysporum V5w2 may not be noticed, and why the plants in Chapter 8 seemed to have recovered at the time of planting. This may also affect the criteria used in qualifying field- grown donor banana plants for F. oxysporum endophytes as healthy or symptomless (see Schuster et al. 1995; Niere et al. 1999; Niere 2001; Dubois et al. 2006a,b). In the screenhouse, R. similis density increased in roots of potted banana plants that had been inoculated with F. oxysporum V5w2 and treated with P-deficient solution (Chapter 7). Densities of R. similis increased in F. oxysporum V5w2-treated banana roots without nutrient stress (Athman et al. 2007). Growth in the form of root biomass and number of leaves was poorer in potted plants that were inoculated with F. oxysporum V5w2 than those without the endophyte when K-deficient solution was applied (Chapter 7).
171 Summarizing discussion
Therefore, deficiencies in P and K may result in negative effects of F. oxysporum V5w2 in banana plants. This deserves further investigation. Plant growth suppression (Chapters 7 and 8), and poor condition of young plants (Chapter 8), may be considered to be some costs related to hosting of endophytes (Heil et al. 2000; Cheplick 2007). Non-pathogenic F. oxysporum V5w2 has been shown to have no relationship with some pathogenic F. oxysporum strains (Vu 2005). Understanding the actual causes for plant growth suppression and increase in nematode densities as recorded in this thesis would contribute to improvement of the endophyte technology.
Knowledge gaps in this thesis First and foremost, it should be established whether the method for inoculation that I used, and which involved exposing undamaged roots to the endophyte inoculum was successful. The fact that I have not effectively analyzed successful inoculation is a major shortcoming of the studies presented here. There are issues that, if addressed, would have facilitated better comparison between experiments in this thesis, and with other studies. The experiments in this thesis mainly addressed the effects of nutrients, microorganisms and mulching, but did not adequately explore the mechanisms of action between F. oxysporum V5w2, the pests and banana plants. The studies in this thesis do not provide information on changes in soil properties that may have occurred due to mulching and fertilizer application. Also, the experiments did not investigate the effects of chemical
172 Chapter 9 changes, which may have occurred due to soil sterilization (Troelstra et al. 2001; De Deyn et al. 2004). Studies in this thesis did not provide a concrete basis for linking positive or negative plant performance to specific soil microbial taxa. Furthermore, information on nutritional quality of banana plants to the endophyte and pests under different treatments is still deficient in this thesis. There is a need to confirm the initial status of tissue culture banana plants with respect to the absence of endophytic microbes. This is the foundation of the endophyte-enhanced tissue culture banana technology (Sikora et al. 2000), but is not indicated in the protocol that I used (Fig. 1.3).
Prospects for implementation of the endophyte-enhanced banana tissue culture technology Knowledge on the potential of non-pathogenic F. oxysporum in controlling nematodes was conceived in the year 1989 (Sikora et al. 2008). Since then, endophyte-enhanced tissue culture banana technology has been made available for farmers’ fields (Pocasangre 2006; Dubois et al. 2006b, Dubois and Coyne 2009). There have been ongoing farmer-based trials for F. oxysporum V5w2 and other endophytes in Uganda among other East African countries, and with some large-scale banana producers in countries like Costa Rica (Dubois et al. 2006b; Pocasangre 2006; Pocasangre et al. 2006). Fusarium oxysporum V5w2 is among endophytes that have been undergoing registration for commercialization as biopesticides in East Africa (Kahangi et al. unpublished; Waithira 2009; Dubois and Coyne 2009). The suppressiveness of F. oxysporum V5w2 against R. similis has
173 Summarizing discussion
been shown before (Sikora et al. 2008; zum Felde 2008, et al. 2009; Paparu et al. 2009b). In this thesis, the biological control potential of F. oxysporum V5w2 against R. similis was investigated in relation to banana plant nutrition, soil sterility and mulching. The studies in this thesis found no consistent evidence for the protection of F. oxysporum V5w2-inoculated plants against root damage by R. similis. Fusarium oxysporum V5w2- inoculated plants even had increased nematode infections (Chapter 7), with reduced growth (Chapters 7 and 8). Sikora et al. (2008) mentioned that there is scarcity of published information on endophytic control of pests using F. oxysporum, as the research field is relatively new, while some work exists in the form of Ph.D. theses or in unpublished form. In the future, studies need to elaborate on other possible mechanisms through which F. oxysporum V5w2 affects nematode infections in banana roots. Policies related to the use of microbial endophytes would need to be developed and strengthened.
Conclusion In this thesis, symptoms of infection by R. similis varied depending on plant nutrition. Fertilizer application helped suppress the activities of C. sordidus and R. similis in banana plants. The soil expressed nematode-suppressive activities that were eliminated by sterilization. Mulch promoted banana production, but enhanced damage to feeder roots. Fusarium oxysporum V5w2 was partly suppressive to C. sordidus in potted banana plants grown in sterilized soil. Also, F. oxysporum V5w2 was suppressive to R. similis in N-deficient potted banana plants that were stunted, but not in plants that
174 Chapter 9 received only water, a complete nutrient solution, or P- and K-deficient solutions. Potted plants that were treated with F. oxysporum V5w2 had poor growth under K-deficiency, with high R. similis density when inoculated with the nematode under P-deficiency. Apart from suppressing root death under N-deficiency, my experiments did not provide other evidence on the ability of F. oxysporum V5w2 to inhibit root damage caused by R. similis. In the field, F. oxysporum V5w2-treated plants expressed a suppressed growth. In-depth understanding of other possible mechanisms involved in the effects of endophytes on plant pests is essential for drawing any deductions and making testable predictions. This remains to be done in future studies. In conclusion, data on the effect of nutrients, soil microorganisms and mulching do not support the transfer of F. oxysporum V5w2-treated banana plants to farmers, because the plants suffer from reduced performance. However, the lack of valuable data on whether the inoculation of plants with F. oxysporum V5w2 was successful is a main impediment to drawing a final conclusion on the effect of the endophyte in my experiments.
Acknowledgements I acknowledge Prof. Arnold van Huis, Prof. Marcel Dicke, Dr. Thomas Dubois, Dr. Piet van Asten and Dr. Edward Muge for reviewing this chapter.
175
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194 Summary
The production of East African highland cooking banana is being hindered by the herbivorous weevil Cosmopolites sordidus and plant-parasitic nematodes especially Radopholus similis. The endophyte-enhanced tissue culture banana technology has been developed for pest management. The technology involves inoculation of tissue culture banana plants with pest- inhibitive endophytes like Fusarium oxysporum V5w2. The endophytic fungus controls pests through induced resistance and antibiosis. The entomopathogenic fungus Beauveria bassiana G41 is being applied as an artificial endophyte against C. sordidus. The endophyte-enhanced tissue culture banana technology has been made available for farmers’ fields. However, information is required on how pest management is affected by interactions between endophyte-enhanced banana plants with soil microbial biotic factors and nutritional abiotic factors (N, P and K), as well as how the relationships are influenced by mulching. The information would be used to validate the transfer of the endophyte-enhanced tissue culture banana plants to farmers. The aim of the research described in this thesis is to provide such information. Chapter 1 focuses on clarifying the endophyte concept and the technology of applying endophytic F. oxysporum V5w2 in relation to banana plants and pests. This issue has been addressed by using the classification of endophytes. Any microbe inside plant tissues is an endophyte, and F. oxysporum V5w2 is classified as non-pathogenic. Beauveria bassiana G41 is classified as an ‘artificial endophyte’ as it could be re-isolated from surface-sterilized banana tissues after inoculation, although the fungus does not form natural relationships with the plants. The
195 Summary chapter describes the endophyte-enhanced tissue banana culture technology. Some key issues in this technology include isolation of endophytes from healthy plants; assuring non-pathogenicity on both host and non-host plants; determining endophyte modes-of-action and persistence; development of inoculation methods; and laboratory, screenhouse, field and on-farm experiments. Chapter 2 is a description of the setup of this thesis, which explains the aims and objectives of the research and how they are addressed in Chapters 3 to 9. In particular, Chapters 4 to 7 are derived from three experiments with potted banana plants that address interactions between R. similis (with or without) and F. oxysporum V5w2 (with or without), as affected by soil microorganisms through comparisons between sterile and non-sterile soil. Simultaneously, the effects of nutrients are studied by comparing plant growth and nematode infection between complete nutrient solution (CNS), only water, or solutions that are deficient in N, P or K. The experiment has yielded mixed results e.g. F. oxysporum V5w2 suppresses, enhances and has no effect on plant growth and R. similis depending on experimental conditions. It is a challenge to unravel under what conditions the biological control potential of F. oxysporum V5w2 against R. similis is successful and what the underlying mechanisms are. For that reason, the studies have been refocused from the biological control subject towards understanding plant-nematode-microbe interactions with soil biotic and abiotic factors. The experimental treatments are organized as follows: data from plants without the endophyte treatment in Chapter 4 are used to address variations in R. similis infection and plant growth in sterile and non-
196 Summary sterile soil, as affected by CNS, water only, or solutions that were deficient in N, P or K; the effect of F. oxysporum V5w2 on R. similis in sterile and non-sterile soil is assessed under uniform nutritional conditions i.e. CNS in Chapter 5, only water or N-deficient solution in Chapter 6, and P- or K- deficient solutions in Chapter 7. Chapter 3 addresses the suitability of banana plants that are inoculated with F. oxysporum V5w2, with or without fertilizer, as hosts for C. sordidus. Fusarium oxysporum V5w2-inoculated plants are less preferred and less damaged by C. sordidus, and these responses are not influenced by fertilizer application. In this chapter, C. sordidus also has low preference for banana plants inoculated with the artificial endophyte B. bassiana G41. Larval C. sordidus that are reared on nutrient-deficient plants are heavier than those from fertilizer-treated plants, which might be due to high levels of defensive or toxic compounds in the fertilizer-treated plants. In Chapter 4, symptoms of banana infection with R. similis vary depending on nutrient treatments and soil sterility. For instance, the nematode treatment is correlated with enhanced plant growth when an N- deficient solution is applied. Also, root necrosis is higher in plants grown under P-deficiency, and lower under K-deficiency, when compared to those treated with complete nutrient solution (CNS). The endophyte has no effect on R. similis when plants are treated with CNS (Chapter 5), or when they receive only water (Chapter 6). Suppression of R. similis density by F. oxysporum V5w2 is achieved in plants that receive N-deficient solution (Chapter 6). However, R. similis density increases when F. oxysporum V5w2-inoculated plants are treated with P-deficient solution (Chapter 7).
197 Summary
Also, inoculation of plants with F. oxysporum V5w2 results in low root biomass and few leaves when K-deficient solution is applied (Chapter 7). Radopholus similis densities are lower in non-sterile than in sterile soil indicating the presence of nematode-suppressive microbes in non-sterile soil (Chapters 4-7). Plant growth is poorer in non-sterile than in sterile soil when N is not applied (Chapters 4-6), but is better when N is present (Chapters 4, 5 and 7), indicating microbial competition for N constrains plant growth. Chapter 8 addresses the compatibility of mulching and F. oxysporum V5w2 for nematode control in field-grown banana plants. Mulch promotes plant growth and bunch size. Contrarily, the endophyte suppresses plant growth and shortens the ripening period for bunches. Less toppling occurs on endophyte-treated plants on mulched plots possibly because the plants are small during growth. Chapter 9 is the summarizing chapter of the thesis. Fertilizer application promotes plant growth and the suppression of C. sordidus and R. similis. Mulch promotes plant growth and bunch size. Treatment of plants with F. oxysporum V5w2 or B. bassiana G41 as single inocula inhibit C. sordidus. Root treatment with F. oxysporum V5w2 as a single inoculum suppresses plant growth, and only inhibits R. similis when plants receive N- deficient solution. In conclusion, data on the effect of nutrients, soil microorganisms and mulching do not support the transfer of F. oxysporum V5w2-treated banana plants to farmers, because the plants suffer from reduced performance. Understanding endophytic mechanisms of action and establishing successful inoculation is necessary for drawing a final valid conclusion.
198 Samenvatting
De productie van de Oost Afrikaanse hoogland banaan wordt verlaagd door de herbivore snuitkever Cosmopolites sordidus en door plantparasiterende nematoden, vooral Radopholus similis. De technologie van bananen weefsel cultuur verrijkt met endofyten is ontwikkeld als een methode ter bestrijding van plagen. In deze technologie wordt een weefselcultuur van bananenplanten geïnoculeerd met plaagremmende endofyten zoals Fusarium oxysporum V5w2. Deze endofyte schimmel bestrijdt een plaag door middel van een geïnduceerde weerstand en antibiosis. De entomopathogene schimmel Beauveria bassiana G41 wordt toegediend als een artificiële endofyt tegen C. sordidus. Weefselcultuur van bananen verrijkt met deze endofyt is ontwikkeld voor toepassing door boeren. Echter, informatie over het effect van de interactie tussen de endofyt-verrijkte bananenplanten, bodem biotische factoren en abiotische factoren (de voedingsstoffen N, P en K) op plaagbestrijding, alsmede de invloed van het gebruik van mulch op deze interacties, is vereist. Deze kennis zou gebruikt moeten worden om de levering van de endofyt-verrijkte bananenplanten aan boeren te valideren. Het doel van het onderzoek beschreven in dit proefschrift is om hier informatie over te krijgen. In Hoofdstuk 1 is het begrip ‘endofyt’ verduidelijkt en de technologie beschreven van het toedienen van de endofyt F. oxysporum V5w2 in het kader van bananenplanten en de bijbehorende plaagorganismen. De classificatie van endofyten vormt daarbij de basis. Elke microbe in plantenweefsel wordt beschouwd als een endofyt, en F. oxysporum V5w2 is ingedeeld als niet pathogeen. Beauveria bassiana G41 is ingedeeld als een ‘kunstmatige endofyt’ doordat het opnieuw geïsoleerd
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kan worden uit oppervlakte-gesteriliseerd bananenweefsel na inoculatie, hoewel de schimmel geen natuurlijke relatie heeft met de plant. Het hoofdstuk beschrijft de endofyt-geïnduceerde bananen weefselcultuur technologie. Belangrijke aspecten van deze technologie zijn de volgende: isolatie van endofyten uit gezonde planten; verzekeren dat het voor zowel waardplanten als niet waardplanten niet pathogeen is; bepalen van de werking en persistentie van endofyten; ontwikkeling van inoculatie methoden; en experimenten in het laboratorium, de plantenkas, het veld en in de praktijk op landbouwbedrijven. Hoofdstuk 2 beschrijft de opzet van het proefschrift. De doelstellingen van het onderzoek worden beschreven, en de manier waarop hiermee omgegaan wordt in Hoofdstuk 3 tot en met 9. Meer specifiek, Hoofdstukken 4 tot en met 7 analyseren drie experimenten met opgepotte bananenplanten, waarbij de interacties tussen R. similis (zonder en met) en F. oxysporum V5w2 (zonder en met) onder invloed van bodem micro- organismen werden onderzocht in zowel steriele als niet-steriele grond. Tegelijkertijd zijn de effecten van bodemnutriënten bestudeerd door de mate van plantengroei en nematodenaantasting te vergelijken tussen een ‘komplete voedingsoplossing’ (CVO), enkel water, en oplossingen met een tekort aan één van de elementen N, P of K. De experimenten gaven tegenstrijdige resultaten. Bijvoorbeeld, afhankelijk van proefomstandigheden worden plantengroei en R. similis door F. oxysporum V5w2 onderdrukt of versterkt, of blijven gelijk. Het is een uitdaging om te bepalen onder welke omstandigheden de bestrijding met F. oxysporum V5w2 van R. similis succes heeft en welke mechanismen daarbij een rol
200 Samenvatting
spelen. Hierdoor werd het onderzoek gericht op het begrijpen van de interacties van plant, nematode en microbe onder wisselende bodem biotische en abiotische factoren. De experimenten zijn als volgt behandeld. Data van planten zonder toediening van de endofyt uit Hoofdstuk 4 zijn gebruikt om variaties te verklaren in zowel aantasting van R. similis als plantengroei in steriele en niet-steriele grond, waar telkens aan zijn toegevoegd: CVO, enkel water, en oplossingen met een tekort aan één van de elementen N, P of K. Het effect van F. oxysporum V5w2 op R. similis in steriele en niet-steriele grond is bepaald onder uniforme condities, i.e. CVO in Hoofdstuk 5, enkel water en oplossing met een tekort aan N in Hoofdstuk 6, en oplossingen met een tekort aan P en K in Hoofdstuk 7. In Hoofdstuk 3 wordt de geschiktheid van bananenplanten als waardplant voor C. sordidus geëvalueerd, wanneer geïnoculeerd met F. oxysporum V5w2 met en zonder toevoeging van meststoffen. Planten geïnoculeerd met F. oxysporum V5w2 worden minder gekozen en zijn dus minder aangetast door C. sordidus. Dit resultaat wordt niet beïnvloed door de toegediende meststoffen. Daarnaast blijkt dat C. sordidus ook weinig voorkeur heeft voor bananenplanten die geïnoculeerd zijn met de artificiële endofyt B. bassiana G41. Larven van C. sordidus die gekweekt zijn op planten, die weinig voedingstoffen hebben gekregen, hebben een lager lichaamsgewicht dan larven gekweekt op planten die bemest zijn. Dit is mogelijk als planten die meststoffen hebben gekregen veel defensieve of toxische stoffen hebben aangemaakt. In Hoofdstuk 4 variëren de symptomen van bananenplanten aangetast door R. similis met de toegediende meststoffen en de steriliteit van
201 Samenvatting
de grond. Bijvoorbeeld wanneer een oplossing met een tekort aan N is toegediend, verbetert de nematode de plantengroei. Daarnaast ontwikkelen planten waaraan een oplossing met een tekort aan P is toegediend meer wortelnecrose, en bij een tekort aan K minder wortelnecrose, wanneer vergeleken met planten waaraan CVO is toegediend. De endofyt heeft geen effect op R. similis wanneer planten CVO hebben gehad (Hoofdstuk 5), of enkel water hebben gekregen (Hoofdstuk 6). Onderdrukking van de dichtheid van R. similis door F. oxysporum V5w2 vindt plaats in planten die een tekort aan N hebben gekregen (Hoofdstuk 6). Echter wanneer Fusarium oxysporum V5w2 geïnoculeerde planten worden behandeld met een voedingsoplossing met een tekort aan P dan neemt de dichtheid van R. similis toe (Hoofdstuk 7). Ook hebben Fusarium oxysporum V5w2 geïnoculeerde planten een lage wortel biomassa en weinig blad bij toediening van een oplossing met een tekort aan K (Hoofdstuk 7). Radopholus similis dichtheden zijn lager in niet-steriele dan in steriele grond wat duidt op de aanwezigheid van nematoden onderdrukkende microben in de niet-steriele grond (Hoofdstukken 4-7). Planten groeien slecht in niet-steriele grond zonder toediening van N (Hoofdstukken 4-6), maar groeien beter wanneer N is toegevoegd (Hoofdstukken 4, 5 en 7). Dit duidt aan dat microbiële concurrentie voor N de plantengroei belemmerd. Hoofdstuk 8 is een veldproef met bananen en gaat over de interactie tussen mulchen en het gebruik van F. oxysporum V5w2 ter bestrijding van nematoden. Het gebruik van mulch bevordert de plantengroei en de grootte van bananentrossen. Daarentegen onderdrukt de endofyt de groei van de planten, terwijl de rijping van de bananen langer duurt. Bananenplanten met
202 Samenvatting
endofyten op velden met mulch vallen minder snel om, mogelijk omdat de planten gedurende de groei kleiner zijn. Hoofdstuk 9 vat het proefschrift samen. Het toedienen van meststoffen bevordert de plantengroei en onderdrukt C. sordidus en R. similis. Ook mulchen bevordert zowel de plantengroei als de trosgrootte. Behandeling van planten met F. oxysporum V5w2 of met B. bassiana G41 geeft een lagere aantasting van C. sordidus. Toedienen van F. oxysporum V5w2 aan wortels onderdrukt de plantengroei, en verhindert R. similis alleen als een voedingsoplossing zonder N wordt toegediend. Onze resultaten van het effect van voedingsstoffen, micro-organismen in de grond en mulching op bananenplanten zijn zodanig dat er geen aanleiding is om op dit moment V5w2-behandelde planten aan boeren aan te bevelen. Het verder begrijpen van de werking van de endofyt en het verkrijgen van een geslaagde inoculatie zijn voorwaarden om een eindconclusie te kunnen trekken.
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Personal history
Dennis M.W. Ochieno was born on December 21st 1975, in Mombasa, Kenya. He started formal education at St. Mary’s Nursery School and Magongo Primary School in Changamwe, Mombasa, between 1981 and 1989. He proceeded to Mundika Secondary School in Busia, Kenya, between 1990 and 1993. While in secondary school, he participated in the Kenyan National Science Congress in 1992 and 1993. In the year 2000, he obtained a B.Ed. (Sci) degree, specializing in Botany and Zoology at Egerton University in Njoro, Kenya. While pursuing the Bachelors degree, he taught Biology and Chemistry at Nyang’ori Secondary School in Vihiga, Kenya. He joined the University of Nairobi and earned a Masters of Science degree in Agricultural Entomology in 2005. His M.Sc. research, which was funded by USAID at the International Centre of Insect Physiology and Ecology (ICIPE) in Nairobi, was entitled ‘Studies of the potential non-target effects of Bacillus thuringiensis Cry1Ab toxin on two stemborer parasitoids Cotesia flavipes Cameron, Xanthopimpla stemmator Thunberg, and the butterfly Acraea eponina Cramer’. While still doing M.Sc. research, he was a member of the GMO Guidelines Project in which he co-authored the book chapter ‘Biodiversity and non-target impacts: a case study of Bt maize in Kenya. In: Hilbeck and Andow (Eds) Environmental Risk Assessment of Genetically Modified Organisms: A Case Study of Bt Maize in Kenya. CABI, Wallingford, UK, pp. 117-185’. Between 2005 and 2006, Ochieno was employed as a consultant to develop research proposals on the control of banana pests using endophytic microorganisms at the International Institute of Tropical Agriculture (IITA) in Namulonge, Uganda. In the year 2006, he was recruited for a sandwich Ph.D. program, which comprised the
205 Personal history work reported in this thesis, at the Laboratory of Entomology, Wageningen University and IITA, Uganda. Within this BMZ-funded Ph.D. research, Ochieno supervised theses of four students registered at Wageningen University and Makerere University. In 2009, Ochieno was appointed honorary lecturer at the College of Biological and Physical Sciences (CBPS), University of Nairobi. Ochieno’s professional interests are in the fields of Education, Integrated Crop and Pest Management, Biotechnology, Biodiversity, Biosafety and Environmental Protection.
206 PE&RC PhD Education Certificate
With the educational activities listed below the PhD candidate has complied with the educational requirements set by the C.T. de Wit Graduate School for Production Ecology and Resource Conservation (PE&RC) which comprises of a minimum total of 32 ECTS (= 22 weeks of activities)
Review of Literature (5.6ECTS) - The role of biotic and abiotic factors on endophyte-plant interactions in integrated pest management (2006)
Writing of Project Proposal (7 ECTS) - Effect of crop management on performance of endophytic Fusarium spp. in tissue culture banana against the banana weevil Cosmopolites sordidus (Germar) and nematodes in Uganda; EPS (2006)
Laboratory Training and Working Visits (4.3 ECTS) - Identification of root-invading microbes in banana plants; Conservation and Sustainable Management of Below-Ground Biodiversity (CSM-BGBD) project, Makerere University (2007) - Analysis of nutrient content in soil and plant material; National Agricultural Research Organization (NARO), Kawanda, Uganda; International Institute of Tropical Agriculture (IITA), Uganda (2008)
Post-Graduate Courses (1.3 ECTS) - Advanced Statistics; PE&RC (2006)
Deficiency, Refresh, Brush-up Courses (13.5 ECTS) - Insect-Plant Interactions; Laboratory of Entomology (2006) - Molecular and Evolutionary Ecology; Laboratory of Genetics (2006) - Basic Statistics; PE&RC (2006)
Competence Strengthening / Skills Courses (0.6ECTS) - PhD Competence Assessment; Wageningen Graduate Schools (2006)
Discussion Groups / Local Seminars and Other Scientific Meetings (7 ECTS) - Plant-Insect Interaction (2009) - PhD Lunch meetings in the Laboratory of Entomology (2006, 2009) - Student discussions in IITA, Uganda (2006-2009)
PE&RC Annual Meetings, Seminars and the PE&RC Weekend (1.5 ECTS) - PE&RC Weekend (2006) - PE&RC Day (2009) - Plant Sciences Seminars (2009)
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International Symposia, Workshops and Conferences (10.2 ECTS) - 13th Symposium on Insect-Plant Relationships; Uppsala, Sweden; poster presented (2007) - The International Endophyte Workshop on Managing Micro-organisms to Enhance Plant Health for Sustainable Banana Production in Eastern Africa; IITA, Namulonge, Uganda; oral presentation (2007) - Banana2008 Conference; Mombasa, Kenya; poster presented (2008)
Supervision of MSc Students (4 students; 1 year) - Olfactory response of Cosmopolites sordidus (Germar) is affected by volatiles emitted by banana plants inoculated with non-pathogenic Fusarium oxysporum and Beauveria bassiana - Effect of nitrogen on banana parasitism by the root-burrowing nematode Radopholus similis - Effect of soil biotic factors on parasitism by Radopholus similis and nitrogen nutrition in bananas - Occurrence of soilborne microbes invading roots of tissue culture bananas inoculated with endophytic Fusarium oxysporum V5w2
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Funding acknowledgements The Ph.D. research reported in this thesis was conducted under the management of Wageningen University and Research Centre (WUR) and the International Institute of Tropical Agriculture (IITA) in Uganda. Thanks to the German Ministry of Economic Cooperation and Development (BMZ) and WUR for funding this work. We thank the Dr. Judith Zwartz Foundation for contributing to the costs of printing this thesis.
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