WP4 Results

Testing germplasm response for integration with EBCAs

The general objective of WP4 is to test the banana germplasm response against plant parasitic nematodes (PPN), Panama Disease (PD) and banana weevil (BW) in combination with beneficial endophytes and biocontrol agents (EBCAs), and to identify the corresponding key genes/molecular pathways induced in the plants during their response. It is, therefore, important to screen different genotypes to identify: a) promiscuous EBCAs, in a compatible interaction with Musaspp. germplasm; b) EBCAs able to proliferate on a given genotype; and c) genes/molecular pathways activated in the host response to pests and pathogen attacks when EBCAs are present. The following specific objectives have been established:

• Test a collection of newly isolated and/or commercially available EBCAs for their potential activity as growth stimulators and biocontrol agents in different banana genotypes and environments (greenhouse, nursery and field). 
• Detect differentially expressed genes in banana plants showing growth stimulation and biocontrol activity by the EBCAs tested. 
• Identify if the growth stimulators and biocontrol agents identified show endophytism and are able to internally colonize banana plant tissues. 
• Analyse changes in the rhizospheric microbiota associated to banana plants inoculated with EBCAs. 

Task 4.1. Gene expression in tolerant/susceptible bananas under biotic stresses. 

In order to find new EBCAs effective against PPN, PD and/or BW in a range of banana cultivars, Real IPM and KU LEUVEN tested commercially available microorganisms on different genotypes and in different environments/conditions (greenhouse, nursery and open field). The microorganisms were provided by Real IPM Company (Kenya) Ltd., and included fungi such as Trichoderma or Glomus spp., as well as bacteria such asBacillusor Azospirillumspp. Their potential as PGPM was tested in banana, as they stimulate growth in other crops acting as biofertilizers and biocontrol agents. In the case of Trichoderma andBacillusspp.,the potential effect in alleviating drought stress (main abiotic factor limiting banana productions, worldwide) was also tested. Preliminary datafrom a field trial on cv Williams indicated that Glomus spp.,Trichoderma,Bacillusand Azospirillum spp.promoted growth in banana plantlets under nursery conditions. Alternatively, Azospirillumsp. likely stimulated growth at early stages in post-inoculation.  

In a second greenhouse assay, Trichodermasp. was inoculated on different banana genotypes grown either in liquid medium (Gran Enana and Yangambi km5) or in soil (Valery, Pequeña Enana and Yangambi Km5). Two furher trials were also carried out in a tropical environment (Dominican Republic) to evaluate the growth promoting effect in cv Williams (Cavendish subgroup, AAA) nursery plants of the nitrogen-fixing bacterium Azospirillum  sp., the arbuscular mycorrhiza fungus Glomussp., the phosphate solubilizersTrichoderma,Bacillus spp.,and a combination of Trichoderma+ Bacillus + Glomus spp.

A combination of available resistance in plants with protection conferred by EBCAs under field conditions may lead to increased, durable and sustainable IPM during farming. The interaction between endophytes and hosts are often (with varying degrees) highly specific and might be explained in a gene-for-gene type of interaction. Mechanisms through which EBCAs reduce disease severity may be through direct (same ecological niche, competition for nutrients and antibiosis) or indirect antagonism (reduced disease severity by induced disease resistance). The endophyte-host-pest association is complex and certain species will activate enzymatic host-plant defence mechanisms following inoculation.

A first greenhouse experiment was initiated at KU LEUVEN in December 2017 using Trichodermaand Bacillusspp., both provided by Real IPM, to explore growth promoting effects. A total of 48in vitroplants of cv Williams were inoculated by root dipping at the moment of transferring from in vitrotubes to pots (Fig. 1). Williams was selected being widely used in Latin America and being sensitive to PPN and BW, while resistant to PD.  

A total of 48in vitroplants from cv Williams were inoculated by root dipping at the moment of transferring from in vitrotubes to pots. Half of them (24 plants) were inoculated with Real Trichoderma at a concentration of 10⁵cfu/mL, while the other half were inoculated with Real Bacillus at 10⁶cfu/mL. Other 48 in vitroplants were used as controls (total number of plants in the experiment = 96). Subsequently, two more inoculations were applied by drenching (5% of pot volume) after 2 weeks and after 1 month. 

As one month is the approx. time required by banana plants to emerge from the in vitrostage, this timing was selected to start a differential irrigation regime. Aim was to test if the inoculated microorganisms could help in alleviating drought stress in banana. A third inoculation by drenching was made with Trichodermasp., ten weeks after starting the differential water supply. Table 1 summarizes the different treatments applied.

Treatment	Application	Microorganism	Irrigation*	N. plants
1	Water + Tween20	Control	A	12
2	Trichoderma	Trichoderma sp.	A	12
3	Water + Tween20	Control	B	12
4	Trichoderma	Trichoderma sp.	B	12
5	Water	Control	A	12
6	Bacillus	Bacillus sp.	A	12
7	Water	Control	B	12
8	Bacillus	Bacillus sp.	B	12

^*A: optimal irrigation (continuous dripping irrigation; 1 drip per pot). B: suboptimal irrigation (dripping irrigation 3 days per week; 1 drip per pot).

Table 1.Treatments applied to test the growth promoting effects of selected Trichodermaand Bacillusspp. on cv Williams plants with differential water supply, under greenhouse conditions.

An overview of plants during the differential water supply is presented in Fig. 2 (complete randomized design). The number of newly formed leaves and the pseudostem height were recorded weekly, for each combination of microorganism-irrigation treatment and during the differential water supply. Additionally, the projected leaf area (canopy) of the plants was measured 5 times during this period (Fig. 3 A-G). 

After 13 weeks of differential irrigation regime, the greenhouse trial was terminated and recording leaf area, number of newly formed leaves, pseudostem height/girth, number of primary roots and fresh weight of the newly formed leaves. Additionally, samples from the different plant tissues (roots, corm, leaves) and from the rhizosphere were collected for gene expression and microbiota analyses, respectively (Fig. 3).

Data from the first greenhouse experiment showed that plants of cv Williams, inoculated with Trichodermasp. and under optimal irrigation regimes, significantly increased the number of leaves, the leaf area and the number of primary roots. 

Subsequently, the data were analysed and a significant increase in leaf area and number of primary roots was found for plants treated with Trichodermasp. under optimal irrigation. By contrast, no significant results were found in any of the treatments for the newly formed leaves or pseudostem growth/girth. No difference was found with Bacillussp. except for the fresh weigh of leaves, with a significant decreased observed for plants under optimal irrigation.

RNA was extracted from the roots collected at the end of treatments 1 and 2. Additionally, primers for 5 banana genes involved in anaerobic metabolism (ADH: alcohol dehydrogenase; PDC: pyruvate decarboxylase; AOX: alternative oxidase; HB: non-symbiotic haemoglobin; ERF1: ethylene response factor 1), 1 gene involved in oxidative stress (SOD: superoxide dismutase) and 2 genes related to endoplasmic reticulum stress (ERO1: endoplasmic reticulum oxidoreductin-1; bZIP17: bZIP transcription factor 17) have been designed/optimized for qRT-PCR analysis. In parallel, the rhizosphere samples collected from treatments 1 and 2 have been processed and sent for metagenomics sequencing to study the microbial composition of the plants with and without Trichoderma sp., under optimal irrigation (Task 4.2). 

Based on the results of experiment 1, two greenhouse tests using Trichoderma (experiments 2 and 3) started in the frame of two MSc projects (see WP10).

Experiment 2 – In October 2018, plants of genotypes Gran Enana and Yangambi km5 growing in liquid medium were inoculated with Real Trichoderma at 10⁵cfu/mL (Fig. 4A). Gran Enana, the most distributed dessert banana cultivar (Cavendish subgroup AAA) has been reported to be sensitive to PPN and BW, while resistant to some Foc lines causing PD. By contrast, Yangambi km5 is a landrace of subgroup Ibota Bota (AAA), reported as resistant to PPN and PD. This experiment was divided in two independent parts:

1. Sub-experiment 1. Designed to: a) follow the plant growth rate of treated vs.control plants over a period of 3 weeks post-inoculation, and b) obtain expression data of banana genes differentially expressed in contrasting genotypes at 3 weeks post-inoculation (8 plants per cultivar and treatment 32 plants in total). The following non-destructive parameters were recorded at the beginning and end of the assay: pseudostem height, leaf area, plant weight, number of newly formed leaves, number of primary roots and root area (Fig. 4B). Additionally, root weight and pseudostem girth/weight were measured at the end and roots and leaves were sampled for gene expression analyses. Currently, the data collected are analysed and root RNA is being extracted. 

Sub-experiment 2. Designed to detect genes differentially expressed in contrasting genotypes at two early time points post-inoculation. It consisted of 5 plants per cultivar, treatment and time point (40 plants in total). Root samples were taken 24h and 48h post-inoculation. Currently, RNA from root tips is being extracted and a subset of genes will be amplified by qRT-PCR. If differential expression is observed, whole transcriptomic analysis (RNA-seq) will be performed.

Experiment 3 – At the beginning of November 2018, a third experiment in greenhouse has been initiated in which in vitroplants from the genotypes ‘Valery’, ‘Pequeña Enana’ and ‘Yangambi Km5’ were inoculated with Real Trichoderma™ and Asperello™ (Trichoderma sp.provided by Biobest Group) at a concentration of 10⁶cfu/mL (Fig. 5). The first inoculation was made by root dipping and drench at the moment of transferring the plants from in vitrotubes to pots. The second inoculation was made by drench 2 weeks later, when new leaves started to appear. Valery has been selected for being the standard susceptible check for PPN and it is also susceptible to PD. Pequeña Enana is the most common cultivar in Canary Islands and it also belongs to the Cavendish subgroup, being susceptible to PPN and BW, while resistant to PD. 

In the test with Valery, 30 plants were inoculated with Real Trichoderma, 30 plants with Asperello and other 30 plants were kept as controls for both treatments (in total 90 plants were used, arranged in a complete randomized design). In the test with ‘Pequeña Enana’ and ‘Yangambi Km5’, 12 plants per genotype were inoculated with Real Trichoderma, 12 plants per genotype with Asperello and other 12 plants per genotype were kept as controls for both treatments (total: 72 plants, in a complete randomized design) (Fig. 5). The same parameters as for exp. 1 are being measured and tissue/rhizospheric samples will be taken at the end of the assay. The test with Valery will be divided in two different time points (to be determined) for final data collection and sampling.

Assessing growth promotion under nursery conditions. The following plant growth promotion trials have been performed in the nursery of Cristal Vitro Dominicana (Dominican Republic) using young Williams plants.  Trial 1 consisted of 8 microbial treatments with PGPM provided by Real IPM-Biobest and the corresponding control (Table 2). In each treatment, 12 biological replicates were tested.

Treatment	Application	Microorganism	Concentration	Dose rate	N. Plants
1	PTA001	Azospirillum sp.	4 · 108 cfu/mL	10 mL/L	12
2	PTA002	Bacillus sp	1 · 1010 cfu/mL	10 mL/L	12
3	PTA003	Bacillus sp	5 · 109 cfu/mL	10 mL/L	12
4	PTA004	Glomus spp.	5 · 107 cfu/L	50 mL/L	12
5	Myc800	Glomus sp	800 spores/g	4.24 g/L	12
6	Asperello	Trichoderma spT34	109 spores/g	1 g/L	12
7	Real Trichoderma	Trichoderma spTRC9	109 spores/g	1 g/L	12
8	PTA 001 PTA 004 Asperello	Azospirillum sp, Glomusspp. andTrichoderma sp. T34		10 ml/L; 50 ml/L; 1 g/L	12
9	Control	Irrigation water			12

Table 2.Treatments applied in the nursery trial 1at Cristal Vitro Dominicana.

Once the plants were transferred to trays, the microbial and control solutions were additionally applied by drenching with 10% of the pot volume. On Aug. 17^th, 11 days after the first application, plants were transferred to 1.5 L bags filled with the same substrate to ensure enough physical space for the growing roots. Upon transplant, a second drench inoculation was done with the same treatments and at the same concentrations as indicated in Table 15. The treatments were applied a third time by drenching on Sept. 5^th, 4 weeks after first inoculation. During the trial, non-destructive parameters were recorded: pseudostem height, leaf emission rate, pseudostem girth and leaf area (data not analysed yet). Fresh/dry shoot weight and fresh/dry root weight were measured at the end of the trial, 10 weeks after the first inoculations (data not analysed yet). Plants from treatment 4 showed a higher pseudostem than the controls in most of the time points recorded. Plants belonging to treatments 1, 2, 3, 6, 7 and 8 showed a higher leaf emission rate than the controls in the last recorded time point. 

In Trial 2, four microbial treatments were compared to the control and each treatment was applied to 10 plants (Table 3). The trial started on Aug. 7^th2018 and the treatments were applied via root bath and drench as described. The plantlets were transferred to 1.5 L bags and were drenched again with a volume of 0.15 L of the microbial solutions or irrigation water. Plants growth after treatments was assessed measuring non-destructive parameters: pseudostem height, pseudostem girth, leaf emission rate, length of the youngest leaf (Fig 6) and leaf area (data not analysed yet). Fresh/dry shoot weight and fresh/dry root weight were measured at the end of the trial, 10 weeks after the first inoculations (data not analysed yet). Preliminary results 9 weeks after starting the trial indicated that Real Trichoderma and Asperello might promote pseudostem growth and increase in lenght of the youngest leaf.

Treatment	Application	Microorganism	Concentration	Dose rate	N. plants
1	PTA 003	Bacillus sp.	5·109 cfu/mL	10 mL/L	10
2	PTA 004	Glomus spp.	5·107 cfu/L	50 mL/L	10
3	Real Trichoderma	Trichoderma sp.TRC9	109 spores/g	1 g/L	10
4	PTA 003,PTA 004 and Asperello	Bacillus sp., Glomus spp. and Trichoderma sp.T34	5·109 cfu/mL,5·107 cfu/L and109 spores/g	10 mL/L,50 mL/L and1 g/L	10
5	Control	Irrigation water	0		10

Table 3.Treatments applied in the nursery trial 2at Cristal Vitro Dominicana.

In order to carry out all the activities of the MUSA project in which KU Leuven is involved, a subcontracting was required for the technical development of specific assays on susceptible and tolerant banana varieties in response to biotic stress, with and without ECBAs (task 4.1). Due to the limited expertise and lack of appropriate facilities for Plant Microbiology and Phytopathology tests, the Laboratory of Tropical Crop Improvement (Professor Rony Swennen, KU Leuven) has requested the Laboratory of Integrated and Urban Plant Pathology (Professor Sébastien Massart, University of Liège), to act as a subcontractor with respect to the following services:

1. Advice on any microbiological aspect in the project.
2. Viability tests and quantification of commercial microorganisms.
3. Isolating beneficial microorganisms from soils, banana rhizosphere and/or banana tissues.
4. Contributing to inoculations of microorganisms on banana plants.
5. Selection and development of beneficial microorganisms towards a commercial product.
6. In vitro and/or greenhouse maintenance of banana plants.
7. Joint publications with KU Leuven. 
8. Joint work planning with KU Leuven.

This subcontracting started on September 2017 and shall continue over a period of 48 months under an agreement already signed by both parties. The Integrated and Urban Plant Pathology Laboratory of Prof. Sébastien Massart has been selected as the result of a process that respected the subcontracting principles of best value for money and avoidance of Conflict of Interest (Art. 13 of the GA). At the moment of this First Report presentation the subcontracting has not been invoiced yet. 

Assessing growth promotion under open field conditions. The field trial was located in Guayubín, Monte Cristi, Dominican Republic (19°35’N 71°24’W) and consisted of 240 banana plants cv Williams distributed in 12 by 20 rows and planted on June 21^st2018 (Fig. 7). The climatic conditions in Guayubín are tropical with a minimal temperature of 19 °C and a maximal temperature of 34 °C. The average precipitation is 693 mm per year and the humid/moist period stretches from April to beginning of December.

Bioformulation PTA 001, which contains the nitrogen fixing bacterium Azospirillum sp. at 4 · 10⁸cfu/mL, was used at a final concentration of 10 mL/L. The plants were inoculated for the first time in the nursery on March 17^th2018, 14 weeks before planting in the field. Subsequently, 4 additional drench applications were performed in the field at 2, 4, 9 and 11 weeks after planting. The treatment was applied to one block of 120 plants out of which 20 plants were selected randomly. Simultaneously, a control treatment was applied to 20 plants planted at the same time in the same field but on another block of 120 plants.

Starting from the 5^thweek after planting, the following parameters were recorded weekly: number of leaves, pseudostem height and pseudostem diameter. Work is in progress.

The outputs considered are summarized as follows:

• Optimized growth of banana plants and optimized protocol for greenhouse, nursery and field inoculation of banana plants with different PGPM.
• Optimized image-based system for the analysis of leaf and root areas in banana plants grown under greenhouse and nursery conditions.
• Set of plant growth parameters recorded in plants treated with different microorganisms under greenhouse, nursery and open-field conditions.
• Root RNA extracted from plants inoculated with Trichoderma asperellumin greenhouse. 
• Developed primers for qRT-PCR analysis of a specific gene set in cDNA obtained from inoculated and control banana plants.
• Microbial DNA extracted from rhizosphere of plants inoculated with Trichoderma asperellumin greenhouse.

The EARTH trail in Costa Rica consists of three banana cultivars that have been already planted in the MUSA field plot as follows: two cultivars of subgroup Cavendish, Grand Naine (AAA) and Williams (AAA), as well as Red Makabu (AAA). The plots are already established for field evaluation of endophytes application for control plant parasitic nematodes (Fig. 8).

Potential of commercial EBCAs. Pot trials were established by IITA to study the potential of commercially available EBCAs for promoting growth and control of Fusarium wilt pathogen race 1 (Foc race 1) of banana. The commercial EBCAs were Bacillussp.. and Trichodermasp., sourced from Real IPM. Greenhouse pot experiments were conducted with tissue culture derived plants of the cultivars Grand Naine (some Fusarium resistant) and Mchare (Fusarium susceptible). Endophytes were tested for their growth promoting attributes in resistant cultivar and disease suppression efficacy against susceptible cultivar. Results revealed that Bacillussp. significantly enhanced growth of banana plants by 42% to 82%. Trichoderma significantly reduced Fusarium wilt symptoms compared to control and Bacillus sp.treatment. Thus, Trichodermahas potential for managing Foc race 1, while Bacillushas the potential to be used for enhancing banana growth. 

Task 4.2 Rhizosphere metagenomics and EBCAs-induced effects on soil microbial communities.

Metagenomic analyses.Using samples collected from banana type Mchare in Tanzania, we investigated the root associated microbiome of healthy and Foc-infected plants. Preliminary results revealed that the root endophytic microbiome in banana is dominated by Proteobacteria, followed by Actinobacteria and Bacteroidetes, irrespective of cultivars and locations. Comparative community analysis of healthy and Foc-infected roots also revealed that PD reduced the abundance of Pseudomonadales and Streptomycetaceae, known for the production of antagonistic compounds against phytopathogens. Further, root microbiome in Foc-infected banana revealed higher abundance of Flavobacteriales and Rhizobiales endophytes, involved in carbohydrate metabolism. 

To identify differences in endophytes populations contributing to maintain asymptomatic plants in cultivated banana fields infected by PD, IITA developed. the following activities:

• Identification of optimal banana-cultivated agroecosystems for endophyte isolation and characterization.
• Use of metagenomics for endophyte isolation and biological characterization to detect plants with higher differences in endophyte populations between PD-symptomatic and PD–asymptomatic plants. 
• Upon identification of diseased and healthy plants with significant difference in microbiota composition, performing metatranscriptomics to identify differences in expression profiles of plant and microorganism between symptomatic and asymptomatic bananas.
• Integration of data obtained from metagenomics (KU LEUVEN), metatranscriptomics (CNR-IITA), and endophytes microbiological isolation (IITA) analyses to identify new genera/species involved in tolerance to PD.

In a first sampling campaign, three agroecosystem have been selected for sampling around Kampala in Uganda. GPS has been used to record the exact position of each sampled plant. In Kawanda, IITA selected 3 symptomatic and 3 asymptomatic plants for each banana cultivar: Silk (AAA) and Sukali Ndiizi (AAB). Plants were grown in a field trial with Foc, selected for the semi-controlled infection condition that guaranteed the presence of the pathogen in both symptomatic and asymptomatic plants.

In Luwero, IITA selected 5 symptomatic and 5 asymptomatic banana plants of cultivar Sukali Ndiizi (AAB). This field was selected because plants were embedded in an agroforestry ecosystem with coffee and cassava plants that might have influenced more variability in the endophytic population.

In Kisoga, 5 symptomatic and 5 asymptomatic banana plants of the cultivar Sukali Ndiizi (AAB) were selected. In this field banana was in monoculture and symptomatic and asymptomatic plants were identified in distinct spots in the field.

Collected samples consisted of: (i) bulk soil, collected nearby roots, (ii) rizosphere, (iii) roots, and (iv) corm. Endophytes have been isolated according to the protocol shared within MUSA project and DNA extracted for high throughput sequencing for fungal and bacterial composition analysis (performed by KU LEUVEN). Molecular data analysis did not reveal significant difference in microbiota composition between diseased and healthy plants. However, some families/genera hosting biocontrol agents were identified only in healthy plants when the analysis was conducted on samples collected from young sucker. Indeed even when collected from heavily infected plants, this tissue did not present necroses, which significantly affect the characteristics of the macrobiota.Based on these preliminary, promising results, IITA organised a second sampling campaign in November 2018 returning to the Luwero field only. The second sampling included  a preliminary metagenomic analysis. In addition, samples from suckers from either Foc infected mats or aymptomatic mats, were collected from asymptomatic plants. Endophytes were isolated according to the protocol developed by IAS CSIC. DNA was extracted, sequencing the 16S rRNA gene for bacteria and ITS for fungi (analyses in progress). Abundance and diversity analyses are in progress. For this sampling, pairs of plants were identified, one symptomatic and one asymptomatic, located very close to each other. Root and corm samples were collected from young suckers from nine healthy and nine diseased plants in total. Samples are currently under investigation for endophytes isolation and DNA extraction for high throughput sequencing of fungal and bacterial species. 

Metagenomics sequence data have been produced for fungi and bacteria from rhizosphere of Pequeña Enana plants and adiacent, uncultivated control soil, proceeding from Tenerife farms sampledin collaboration by Coplaca and CNR (Feb. 2018). 

SARI with UNEXE progress in WP 4 (Testing germplasm response for integration with EBCAs) was as follows:

• Established local collaborations in Ethiopia to carry out a comprehensive diagnostic study on nematodes of enset with the College of Agriculture and Veterinary Medicine at Jimma.
• Trained two researchers with field sample management and extraction techniques.
• These activities prepared partners to carry out field sample collections and diagnostic studies on nematodes and Fusarium on enset.
• Samples will allow isolation and characterization of EBCAs.