The role of soil types on the relation between individual soil properties and Fusarium wilt expression in 'Gros Michel' bananas

Aims This study looks whether the response of soil management (liming and nitrogen fertilization) on the incidence of Fusarium wilt (Foc Race 1) in Gros Michel banana (Musa AAA) is inuenced by soil types. Methods The effect of inoculation with Foc Race 1 was studied in a factorial greenhouse trial with eight representative soil types of the Costa Rican banana region, two pH levels; and three levels of N-fertilization. After an 8-week period, plant biomass, leaf area, and a disease index were measured. Results There were signicant effects of soil pH and N, and their interactions on disease expression. Low pH levels and high N-fertilization increased the disease expression. The response to changes in soil pH and N-fertilization differed considerably between the different soil types. Conclusions Although soil pH and N inuence Fusarium wilt in banana, each soil type differs in its response to these soil properties. This complicates the development of standard soil management strategies in terms of e.g., N-fertilization and liming to mitigate or ght the disease. the natural to the an erratic behavior of the to


Introduction
Fusarium wilt, also known as 'Panama disease' (caused by the soil-borne fungus Fusarium oxysporum f. sp. cubense or Foc), is one of the most critical diseases affecting banana production. Foc Race 1 devastated the subgroup Gros Michel (Musa AAA), which was the main cultivar exported from Latin America and the Caribbean (LAC) during the rst half of the 20 th century (Ploetz and Churchill 2011;Pocasangre et al. 2017;Dita et al. 2018;Magdama et al. 2020). Foc Race 1 remains a serious problem in small-scale production systems in LAC where the speci c traits of the Gros Michel banana are preferred (Pocasangre et al. 2011). The gradual shift of the production systems to the cultivars of the subgroup Cavendish (Musa AAA), which are resistant to Foc Race 1, was a temporary solution to the problem (Harper 1950;Stover 1961Stover , 1962Ploetz 1990;Perez-Vicente 2004). A new, more aggressive strain of the fungus denominated Foc Tropical Race 4 (TR4) is spreading over the world and has recently been reported in LAC (García-Bastidas et al. 2020). Most of the varieties produced in LAC, including the Gros Michel and the Cavendish subgroups, are susceptible to Foc TR4. The spread of Foc TR4 in LAC would have a tremendous impact given the economic and social importance of banana production in the region (Aurore et al. 2009;Pocasangre et al. 2011;Dita et al. 2013).
Conventional control options such as fungicides, replanting or crop rotation are ineffective in controlling or eradicating the disease (Ploetz 2006(Ploetz , 2015Ordoñez et al. 2015). Other alternatives, such as the evaluation of partially resistant cultivars or breeding new resistant cultivars (Su et al. 1986;Hwang and Ko 2004;Dale et al. 2017), can take a long time to be available for practical implementation. In the short run, it is important to develop a control package that allows farmers to face the disease.
Soil management in agriculture mainly focuses on crop production and rarely considers crop disease control. However, speci c soil conditions can suppress diseases in agricultural crops (Janvier et al. 2007). Managing soil properties can i) in uence the soil microbiome and as a result change disease pressure or ii) in uence the crop nutritional status and change the crop's predisposition to diseases (Dordas 2008;Ghorbani et al. 2008;Huber et al. 2012). Already in 1946, there were reports that liming, fertilization, and crop rotation may be a "cure" to Fusarium Wilt in Banana (Taylor (1946) cited by Jones and Morrison, 1952).
Studies also included ooding of infected areas to aim for soil disinfection or a reduction of the fungus population (Stover 1961). However, through the production of chlamydospores Foc can survive extreme conditions like the anaerobic conditions under ooding (Ploetz 2015). Recently, ooding and irrigation are increasingly being attributed to also increase the spread of the disease (Salacinas 2019). After the shift from Gros Michel to the resistant Cavendish cultivars, research on Fusarium wilt control was limited for more than 30 years. However, since reports of the new and more aggressive strain Foc TR4 (Stover 1986) were published, research on controlling this disease came back. Studies show that Foc Race 1 and Foc TR4 respond in a similar way and that soil properties play an important role in conducing or suppressing the fungus in banana (Domínguez et al. 2008;Orr and Nelson 2018;Segura et al. 2021). However, the results in the literature are found to be inconsistent. This seriously hampers the translation of research results into operational management recommendations. Bananas are grown under a wide variety of agro-ecological conditions (Jaramillo and Vásquez 1990;Stoorvogel and Segura 2018). One possible reason for these inconsistencies could be that the interactions between Fusarium wilt and soil properties differ with agro-ecological conditions. Soil types have long been known to play a role in this relationship (Stotzky et al. 1961).
This study aims to evaluate the role that soil types play on the effect of liming and N-fertilization on the incidence of Fusarium Wilt. Eight representative soil types from the Costa Rican banana region are evaluated studying the incidence of Fusarium wilt by Foc Race 1 in Gros Michel bananas in a large greenhouse experiment. The results may help to better identify the role of soil types in explaining the inconsistencies in results and to support the development soil management strategies to reduce the impact of Fusarium wilt race 1 and help to identify research strategies to similarly develop strategies to control TR4.

Materials And Methods
Banana production in Costa Rica is concentrated in the perhumid Atlantic zone (Figure 1). Soil conditions vary considerably in the area. Soil types were selected during a survey in the banana regions and they represent the variety that was found in the region (Klinkert 2014). The survey included the Caribbean lowlands, which include over 40,000 has of large intensive production of Cavendish cultivars for export (Segura et al., 2015). In addition, the Turrialba region, which has a more extensive production of Gros Michel cultivars for local markets (Ramirez et al. 2010), was incorporated into the study. Soils in the Caribbean lowlands are highly variable (Lopez and Solís 1991; Segura et al. 2015). Soils to the east of the Reventazón river ( Figure 1) are predominantly sedimentary with a high clay content and high fertility. Soils to the west of the Reventazón river originate from volcanic ashes with a low clay content and medium fertility. Soils in the Turrialba region are deep, well-drained, tropical red soils with a high clay percentage and medium fertility (Dijkshoorn et al. 2005). The climate is tropical and humid with an average annual rainfall of 3000-3500 mm distributed throughout the year. The experiment was performed in a greenhouse at the experimental station of CORBANA in La Rita (132 m.a.s.l., 10°15'54' latitude N, 83°46'26'' longitude W, maximum temperature of 35 • C and minimum temperature of 17 • C, average temperature of 28 • C with an 85% relative humidity and approx. 12 h of daylight).
The factorial design included 8 soil types x 2 levels of Foc Race 1(with and without) inoculation x 2 levels of soil pH x 3 levels of N-fertilization x 3 replications resulting in 288 pots. Two contrasting soil pH levels were tested: 1) pH low with a pH of 5.2 or lower, and 2) pH high with a pH equal to or higher than 6.0. Soil pH was adjusted to these target levels by applying a hydrochloric acid solution (10% HCl) to decrease pH or lime (CaCO 3 ) to increase soil pH. Soil pH was adapted before any other treatment were applied. In each case, the acid or alkaline units of solution required to achieve the lower and the higher pH were calculated before liming and/or acidifying. Soil pH was analyzed before each treatment and eight days after the liming or the acidi cation treatments (where they prodeed). In the low pH treatment, pH levels between 4.0 and 5.1 were measured and in the high pH treatment, pH levels between 6.2 and 6.8 were measured.
Hardened, approximately 3-month-old, tissue culture banana plants (Musa AAA, subgroup Gros Michel) were used in the experiment. The plants grew in a standard potting mix before the experiment. Banana plants in young stages from tissue culture, such as the ones used in the experiment, are more sensitive to Foc infestation (Brake et al. 1995). This condition ensured the plant's response to the disease according to the treatments. Two levels of Foc Race 1 inoculation were achieved by root dipping (Dita et al. 2010;García-Bastidas et al. 2014Ordoñez et al. 2016): 1) In 0 : a control, 30 minutes in clean water, and 2) In 1 : the inoculated group, 30 minutes in a solution of water with 10 6 conidia mL -1 of Foc Race 1. The fungus strain was collected from Costa Rican soils, tested, and cultivated by CORBANA's Center of Biological Control. It should be noted that plants in In 0 could still be infected with Foc Race 1 present in the soils. It is widely accepted that Costa Rican banana soils are infested with Foc Race 1. As the soil samples were not sterilized before the experiment in order to not disturb the soil microbiome, the plants in the control are exposed to Foc Race 1 (typically at low concentrations). Immediately after the inoculation, plants were separately planted in 2 L pots (one plant per pot). Three levels of N doses were achieved through weekly differentiated N-fertilization with ammonium nitrate (AN, 33.5% N): N low with no N addition relying on natural N in the soil; 2) N med with 0.08 N g plant -1 week -1 supplied through 0.24 g of AN plant -1 week -1 ; and 3) N high with 0.25 N g plant -1 week -1 supplied through 0.75 g of AN plant week -1 . These N doses were respectively achieved through applications of 300 mL of solutions of AN in water with concentrations of respectively 0.00 g L -1 N, 0.14 g L -1 N and 0.43 g L -1 N, two times week -1 . N med emulated the average N requirement of plants during the rst 10 weeks after planting in real eld conditions. No other nutrients or agro-chemicals (e.g., fungicides, insecticides, etc.) were applied to the plants.
The experimental period was 8 weeks long and at the end of this period, total (above ground plus roots) fresh biomass (g plant -1 ), the plant diameter at the base and the foliar area were measured. As the three variables were highly correlated to each other (> 95%), the data analysis was only carried out on the basis of the fresh biomass. In addition, a non-intrusive way to measure the disease according to the management of the soil properties was following the development of the wilting. The disease index (DI) was obtained adapting the McKinney's formula (McKinney 1923) that was also used by Haddad et al. (2018) and Rocha et al. (2020) in the same way. However, in this case, it was based on the number of sick plants and the wilted leaves: DI(%)= 100.∑(f/n)·(v/x), where; f = number of sick plants; n = total of plants; v = number of leaves with symptoms; and x = total number of leaves (with symptoms and healthy). The presence of the typical symptoms of the wilting of the leaves in previously inoculated plants is reported as a valid element to corroborate the presence of the disease in bananas (Dita et al. 2010;García-Bastidas et al. 2014Hung et al. 2018). Plant biomass from In 0 and In 1 and the wilting per plant data were analyzed using a factorial analysis of variance, which considered involved factors and their interactions: soil type, inoculation, soil pH and N for biomass, and only soil type, pH and N for wilting per plant. The differences between factors were evaluated through a Tukey's analysis.

Results
Natural effect of the soil type and soil pH in not inoculated plants The control group (In 0 ) showed the effect of the soil type and the pH and N management on non (Figure 2). There were considerable differences in the mean biomass per plant according to soil type (P < 0.001). Bananas grown on soils from the West and Turrialba showed the best mean performance with the plants grown on pH high being signi cantly (P < 0.001) larger than plants grown on pH low . However, two soils gave more biomass in plants from pH low . The N dose did not signi cantly affect the biomass over the eight soils (P ≥ 0.730). The effect of N fertilization differed per soil type where in some cases the increased nitrogen levels resulted in an improved performance but in other cases there was a decline in the performance with the increases in Nitrogen. It is likely that these differences are related to the residual N in the original soil samples.

Soil pH and N interactions and plant biomass and disease index (DI)
Inoculation with Foc reduced plant biomass. Biomass in In 1 was signi cantly lower than biomass in In 0 for almost all the soil types and pH levels ( Table 2) The effect of the inoculation expressed as a decline in plant biomass was higher in pH low (61.6%) in contrast to pH high (50.8%). However, pH differences resulted in a very different response to the inoculation of the disease for the different soil types as presented in Figure 3. The gure shows that for most soils a pH increase led to a greater biomass for both the control and the inoculated group. However, the direction and the length of the arrows differ indicating that there is a considerable effect of the soil.
Inoculation led to a signi cant decline in almost all soil -N dose combinations (Table 3). With increasing N-fertilization, typically, the decline in biomass due to inoculation increased. In addition, the biomass according to the interactions of the inoculation with the soil type, the pH level and the N dose was also signi cant. A higher effect of the interaction of the soil and the inoculation was evident in all soils. The response of the Figure 4 shows a sample of plants grown in soils from the three regions. The detrimental effect of the interaction between pH low and inoculation was evident in plants from the three regions. In spite of following the trend of a higher biomass in pH high , the mean biomass in In 1 was contrastingly lower against the control for both pH levels. The interaction of the inoculation and the N dose was expressed in different trends according the soil type and pH levels and the average biomass ( Figure 5). The lower biomass in the N high and pH low . From the possible combinations, the higher wilting took place in N high and pH high .
The single effect of soil pH was signi cant in the DI expression with a higher average of wilting in pH high . The higher DI is offset by the increased performance of the plant at pH high still leading to a net bene t of the increased pH. The DI according the N doses was not signi cant, and its trend was more erratic. Plants from pH high remained more biomass and expressed a lower DI. The interaction pH x N was signi cant (P≤ 0.0040) in the DI and average biomass ( Figure 6). There was a lower biomass in N high and pH low . From the possible combinations, more wilting took place in N high and pH high . The single effect of soil pH was signi cant with a higher DI in pH high . The DI according to the N doses was not signi cant and its trend was more erratic. Plants from pH high maintained more biomass and expressed a lower DI.

Discussion
Soil type were found to have a strong effect on plant performance under natural (not inoculated) and inoculated conditions. Losses in biomass in In 1 can be attributed to the detrimental effect of Fusarium wilt on the plants. Due to the incidence, biomass in the inoculated plants was lower because of the loss of the tissue it produced in the plant. Soil pH high increased the biomass per plant for both In 0 and In 1 treatments. Besides, pH x inoculation interactions on the effect of the disease was stronger in pH low . This they could imply a direct (Almeida et al. 2018), indirect effect, or both, of soil pH on the incidence of the disease. The role of pH as an indicator of soil health in banana and its in uence in soil suppressiveness is known (Pattison et al. 2008;Geense et al. 2015;Segura et al. 2015). Low soil pH (less than 5.2) apparently can stimulate pathogen activity in the soil due to a detrimental effect on soil diversity. However, this was not evaluated in this trial. The lower pH also limits plant nutrient and water uptake (White 2012). This effect can increase banana predisposition to diseases led both by a higher Foc activity and a limited capacity to perform physiological processes against the infestation.
Despite the differences according to the interaction of soil type and inoculation, and the general effect of pH on plant response, the detrimental interaction of lower pH x inoculation was higher in various soil types. The highest effect of the disease expressed as a lower biomass found in the eastern area could be due to the speci c characteristics of these. Although all of them have a high fertility, plants from those soils were more sensible to the disease, especially in pH low .
The soil type as a package can play a natural role in plant status and it can de ne the plant's predisposition to the disease. Previous reports indicated that chemical and physical soil conditions would be linked to soil conduciveness of Fusarium wilt (Scher 1980;Domínguez et al. 2008). However, analyzing the interaction of soil properties and the disease in one single soil can lead to misunderstandings and inconsistent conclusions. Interactions of the soil type with pH, N and other abiotic conditions, such as the higher clay content, Fe and Mn, for instance, would be involved in the plant's response. This knowledge is crucial when considering soil management to deal with or control Fusarium wilt in banana.
The plant's response to the disease can be ruled by the particular ecological condition of each soil. Soils with a lower concentration of SOM in the eastern region, for instance, showed the highest detrimental effect of the disease. In the western region, plant response was erratic according to the inoculation. In fact, the best plant performance under infected and pH low conditions took place with the highest soil SOM (S6, S7 and S8). Plants in pH low were more predisposed to Fusarium wilt in all tested soils. Results allow us to de ne different degrees of risk to the disease, according to the study regions. However even in a same banana region, the magnitude of the response to the disease according to the management of pH and N differed depending on the soil type. Nevertheless, practices such as maintaining a higher pH and increasing SOM through management appear to be preventive measures that can decrease plant predisposition to the disease. This seems to be a standard recommendation for all banana locations could be complex or not applicable. With this information, strategies based on soil management to prevent and deal with Foc can be implemented, in this case speci cally for the Costa Rican reality.
The interaction of soil type with pH and N management in the disease expression should be studied thoroughly. How and where this interaction is more signi cant in the disease can de ne the strategy that should be implemented in crop management. Soil pH low and N high predisposed the plant to be more easily infected and more affected by the disease. A higher level of wilting was found with the N application in both pH levels and it was more signi cant in pH low . It appears that soil pH is a primary factor in the plant's predisposition to the disease in most of the studied soil types. N inputs have important impacts in soil conditions, but the plant's response to the disease was more erratic according to this soil property. At the same time, N ammonia sources are recognized as a main cause for a drop in pH in soils from agricultural lands. Therefore, an integral management of soil properties to alleviate or prevent Fusarium wilt should include pH management and choosing less acidity N sources. In addition, it is necessary to include a soil N analysis in order to de ne the N recommendation for banana plantations.
More integral soil conditions can be playing a role in plant response to the disease. Furthermore, the role that the soil type played in the plant's response to de disease was evident. Although it agrees with the response in terms of suppression or conduction of the disease according to the soil type in Australia (Bowen et al. 2019), the results that we found allowed us to see a complex interaction of the soil type, its properties and the incidence of the disease. The in uence of speci c soil properties linked to the soil type (chemical, physical and microbiological) in each type of soil or each banana region can be part of the scenario of the natural banana's response to the disease. This could be the reason why previous studies have shown an erratic behavior of the disease according to soil management. Probably these results could depend on the region where plantations are established in the different banana locations in Costa Rica. The speci c study of at least soil type and its conditions on banana predisposition to Fusarium wilt in each location or banana region is necessary to better understand the role of soil conditions in Fusarium wilt in bananas. Even ecological and environmental aspects of each region can be playing a role in the incidence of the disease.

Conclusions
Soil properties like pH and N play an important role in banana predisposition to Fusarium wilt and in the disease incidence. However, this relation is strongly in uenced by soil type, i.e., other properties. This complexity clearly hampers the development of uniform management recommendations. Nevertheless, general trends are found. An increase in soil pH supports, in almost all cases, the suppression of Fusarium wilt. For the development of more speci c soil management strategies, it appears to be necessary to view the soil as an integral system of physical, chemical, and biological properties rather than looking at individual soil properties with their thresholds. This complexity could be the cause for the inconsistencies that were found in the literature with respect to the role of soil properties in suppressing or conducing Fusarium wilt in banana.

Declarations
Ethical statement: This manuscript is not submitted to another journal. The manuscript is original, and it is not published elsewhere partially or in full, in any form or language. Besides, it does not concern an expansion of previous work. The study is not split up into several parts to increase the quantity of submissions and submitted to various journals or to one journal over time. Results are presented clearly, honestly, and without fabrication, falsi cation, or inappropriate data manipulation. Data was collected from the greenhouse experiment and managed with statistical software with total honestly and transparence. No data, text, or theories by others are presented as if they were the author's own. All collected data, and the performed analysis are available (as a supplementary le). Proper acknowledgements to other works are given. This piece of work respects third parties' rights such as copyright and/or moral rights Author's contribution statement: All the authors took part in conceptualization of the research and editing the manuscript and consent its publication. RS and JAS performed the greenhouse experiment in Costa Rica. RS and JJS performed the results analysis and RS wrote the manuscript. JSS and JS made important inputs to improve the nal version of the manuscript.