Microbial-Based Biofungicides Mitigate the Damage Caused by Fusarium oxysporum f. sp. cubense Race 1 and Improve the Physiological Performance in Banana.

Fusarium wilt of banana (FWB) is the most limiting disease in this crop. The phytosanitary emergency caused by FWB since 2019 in Colombia has required the development of ecofriendly control methods. The aim of this study was to test the effectiveness of microbial-based biofungicides against FWB caused by Fusarium oxysporum f. sp. cubense race 1 (Foc R1) and correlate such effect with plant physiological parameters. Five Trichoderma (T1 to T4 and T9) and four Bacillus (T5 to T8)-based biofungicides were evaluated in pot experiments. In vitro, dual confrontation tests were also carried out to test whether the in vitro effects on Foc growth were consistent with the in vivo effects. While Trichoderma-based T3, T4, and T9, and Bacillus-based T8, significantly reduced the growth of Foc R1 in vitro, Trichoderma-based T1, T3, T4, and T9 temporarily reduced the Foc population in the soil. However, the incidence progress of FWB was significantly reduced by Bacterial-based T7 (74% efficacy) and Trichoderma-based T2 (50% efficacy). The molecular analysis showed that T7 prevented the inner tissue colonization by Foc R1 in 80% of inoculated plants. The T2, T4, T7, and T9 treatments mitigated the negative effects caused by Foc R1 on plant physiology and growth. Our data allowed us to identify three promising treatments to control FWB, reducing the progress of the disease, delaying the colonization of inner tissue, and mitigating physiological damages. Further studies should be addressed to determine the modes of action of the biocontrol agents against Foc and validate the utilization in the field.

Antagonism of volatile organic compounds of the Bacillus sp. against Fusarium kalimantanense.

Fusarium kalimantanense is a genetic lineage of Fusarium oxysporum f. sp. cubense (Foc) and belongs to the Fusarium oxysporum species complex (FOSC). This pathogen is a causative agent of Panama disease, an infection that has caused damage to the banana crop worldwide. Bacillus sp. (LPPC170) showed preliminary antagonist activity against F. kalimantanense (LPPC130) in vitro tests from the cultivation of axenic culture and co-culture with inhibition of mycelial growth of phytopathogen of 41.23%. According to these findings, volatile organic compounds (VOCs) emitted from Bacillus sp. were obtained by solid-phase microextraction and identified by gas chromatography coupled with a mass spectrometer (GC-MS). The multivariate data analysis tool (PLS-DA and Heatmap) identified short-chain organic acids as the main antagonistic VOCs responsible for inhibiting the mycelial growth of LPPC130. Acetic acid, propanoic acid, butanoic acid, valeric acid, and isovaleric acid exhibited a strong inhibitory effect on the mycelial growth of LPPC130, with inhibition of 20.68%, 33.30%, 26.87%, 43.71%, and 53.10%, respectively. Scanning electron microscopy revealed that VOCs caused damage to the vegetative and reproductive structures of the fungus. These results suggest Bacillus LPPC170 as an excellent biocontrol tool against the phytopathogen causative agents of Panama disease.

The Population of Fusarium oxysporum f. sp. cubense in Brazil Is Not Structured by Vegetative Compatibility Group or by Geographic Origin.

Fusarium wilt, caused by the soilborne fungus Fusarium oxysporum f. sp. cubense (Foc), is considered one of the most destructive diseases of bananas in Brazil. In this study, a collection of 194 monosporic isolates from several banana-producing regions located in different climatic zones along a south-to-north transect in Brazil was formed to assess the genetic structure of the population of Foc. The isolates underwent pathogenicity tests, PCR diagnosis for the detection of tropical race 4, and screening of SIX homolog genes that produce putative effector proteins. The vegetative compatibility group (VCG) of 119 isolates was determined by pairing against 17 internationally known VCG-tester strains. A group of 158 isolates was selected for simple sequence repeat (SSR) genotyping. There was moderate diversity of Foc in Brazil. Eight VCGs were identified: 0120, 0122, 0124, 0125, 0128, 01215, 01220, and 01222, of which 78% of isolates belong to a single VCG, whereas 22% of isolates are assigned to multiple VCGs, belonging to complexes of VCGs. The distribution of VCGs is uneven and independent of the banana genotype. The isolates of a VCG shared a similar profile of SIX homologs, but there was no association with geographic region. Four SSR loci were polymorphic, and, on average, 7.5 alleles were detected per locus. Thirty-five multilocus genotypes (MLGs) were identified. There was no association between VCG and MLGs, and no genetic structure of the population of Foc in Brazil was detected.

Incidence, Spatial Pattern and Temporal Progress of Fusarium Wilt of Bananas.

The effective management of Fusarium wilt of bananas (FWB) depends on the knowledge of the disease dynamics in time and space. The objectives of this work were: to estimate disease intensity and impact, and to investigate the spatial and temporal dynamics of FWB. Fields planted with Silk (n = 10), Pome (n = 17), or Cavendish (n = 3) banana subgroups were surveyed in Brazil, totaling 95 ha. In each field, all plants were visually assessed, and diseased plants were georeferenced. The incidence of FWB and the impact of the disease on the yield on a regional scale were estimated. Spatial patterns were analyzed using quadrat- and distance-based methods. FWB incidence ranged from 0.09% to 41.42%, being higher in Silk fields (median = 14.26%). Impacts of epidemics on yield ranged from 18.4 to 8192.5 kg ha-1 year-1, with an average of 1856.7 kg ha-1 year-1. The higher economic impact of the disease was observed on Silk cultivar with an average loss of USD 1974.2 ha-1 year-1. Overall, estimated losses increased on average by USD 109.8 ha-1 year-1 at each 1% of incidence. Aggregation of FWB was detected by all analytical methods in 13 fields (1 of Cavendish, 11 of Pome, and 1 of Silk). In the other 17 fields, at least one analytical method did not reject the null hypothesis of randomness. One field (5 ha), composed of six plots, was selected for spatial and temporal studies during two years with bi-monthly assessments. A sigmoidal curve represented the FWB progress and the Gompertz model best-fitted disease progress. The level of aggregation varied over time, and evidence of secondary infection to neighboring and distant plants was detected. FWB is a widespread problem in Brazil and yield losses can be of high magnitude. Epidemiology-based management strategies can now be better established.

Weevil Borers Affect the Spatio-Temporal Dynamics of Banana Fusarium Wilt.

Dispersal of propagules of a pathogen has remarkable effects on the development of epidemics. Previous studies suggested that insect pests play a role in the development of Fusarium wilt (FW) epidemics in banana fields. We provided complementary evidence for the involvement of two insect pests of banana, the weevil borer (Cosmopolites sordidus L., WB) and the false weevil borer (Metamasius hemipterus L., FWB), in the dispersal of Fusarium oxysporum f. sp. cubense (Foc) using a comparative epidemiology approach under field conditions. Two banana plots located in a field with historical records of FW epidemics were used; one was managed with Beauveria bassiana to reduce the population of weevils, and the other was left without B. bassiana applications. The number of WB and FWB was monitored biweekly and the FW incidence was quantified bimonthly during two years. The population of WB and the incidence (6.7%) of FW in the plot managed with B. bassiana were lower than in the plot left unmanaged (13%). The monomolecular model best fitted the FW disease progress data, and as expected, the average estimated disease progress rate was lower in the plot managed with the entomopathogenic fungus (r = 0.002) compared to the unmanaged plot (r = 0.006). Aggregation of FW was higher in the field with WB management. WB affected the spatial and temporal dynamics of FW epidemics under field conditions. Management of the insects may reduce yield loss due to FW.

Bioassay on banana rhizome discs for selecting bacteria antagonistic to Fusarium oxysporum f. sp. cubense / Bioensaio em discos de rizomas de bananeiras para a seleção de bactérias antagonistas a Fusarium oxysporum f. sp. cubense

Detached plant organs are alternative materials to in vitro tests for selecting biocontrol agents. On the other hand, the use of scales to quantify injured areas can generate inconsistent results. Rhizospheric and endophytic bacteria were selected as growth inhibitors of Fusarium oxysporum f. sp. cubense (Foc), the causal agent of Panama disease of banana. For this, rhizome discs were treated with 200 µL of antagonist suspension (109 CFU mL−¹) and inoculated with the pathogen. The material was placed in plastic gerbox boxes and incubated in BOD at 25 °C for 12 days. Afterward, the area of rhizome discs (mm²) colonized by Foc was quantified by digital images. The assay was set up in a completely randomized design, with four replications and three discs per replication. The control consisted of untreated and inoculated discs. The results showed the efficiency of this method in selecting the biological control agent, as the 26 isolates were group into five different clusters, with isolates belonging to four of these groups (from '1' to '4') being able to reduce Foc colonization. Isolates 520EB, 993EB, and 531EB had the highest potential for inhibition, with areas of 343.3, 344.1, 364.8 mm², respectively, promoting inhibition ranging from 53 to 56 % of the colonized area compared to the control (782.6 mm²).(AU)

O emprego de órgãos vegetais destacados é uma alternativa aos testes in vitro para seleção de agentes de biocontrole. Por outro lado, o uso de escalas para quantificação de área lesionada pode gerar resultados inconsistentes. Bactérias rizosféricas e endofíticas foram selecionadas como inibidoras do crescimento de Fusarium oxysporum f. sp. cubense (Foc), agente causal do mal-do-Panamá da bananeira. Para tanto, discos de rizoma foram tratados com 200 µL de suspensão dos antagonistas (109 UFC mL-¹) e inoculados com patógeno. O material foi disposto em caixas plásticas tipo gerbox e incubado em BOD a 25 ºC, por doze dias. Após, a área dos discos de rizoma (mm²) colonizada por Foc dos discos foi quantificada por meio de imagens digitais. O ensaio foi montado em delineamento inteiramente casualizado, com quatro repetições e três discos por repetição. Discos não tratados e inoculados compuseram o controle. Com base nos resultados obtidos, foi possível verificar a eficiência do método na seleção de agente de controle biológico, uma vez que foi possível o agrupamento dos 26 isolados utilizados em cinco clústeres distintos, sendo que isolados pertencentes a quatro destes grupos (do '1' a '4') foram capazes de proporcionar a redução da colonização por Foc. Os isolados com maior potencial de inibição foram520EB, 993EB e 531EB com 343,3, 344,1, 364,8 mm2, respectivamente, promovendo inibição variando de 53 a 56 % da área colonizada quando comparados com ao controle (782,6 mm²).(AU)

Bioassay on banana rhizome discs for selecting bacteria antagonistic to Fusarium oxysporum f. sp. cubense / Bioensaio em discos de rizomas de bananeiras para a seleção de bactérias antagonistas a Fusarium oxysporum f. sp. cubense

Detached plant organs are alternative materials to in vitro tests for selecting biocontrol agents. On the other hand, the use of scales to quantify injured areas can generate inconsistent results. Rhizospheric and endophytic bacteria were selected as growth inhibitors of Fusarium oxysporum f. sp. cubense (Foc), the causal agent of Panama disease of banana. For this, rhizome discs were treated with 200 µL of antagonist suspension (109 CFU mL−¹) and inoculated with the pathogen. The material was placed in plastic gerbox boxes and incubated in BOD at 25 °C for 12 days. Afterward, the area of rhizome discs (mm²) colonized by Foc was quantified by digital images. The assay was set up in a completely randomized design, with four replications and three discs per replication. The control consisted of untreated and inoculated discs. The results showed the efficiency of this method in selecting the biological control agent, as the 26 isolates were group into five different clusters, with isolates belonging to four of these groups (from '1' to '4') being able to reduce Foc colonization. Isolates 520EB, 993EB, and 531EB had the highest potential for inhibition, with areas of 343.3, 344.1, 364.8 mm², respectively, promoting inhibition ranging from 53 to 56 % of the colonized area compared to the control (782.6 mm²).

O emprego de órgãos vegetais destacados é uma alternativa aos testes in vitro para seleção de agentes de biocontrole. Por outro lado, o uso de escalas para quantificação de área lesionada pode gerar resultados inconsistentes. Bactérias rizosféricas e endofíticas foram selecionadas como inibidoras do crescimento de Fusarium oxysporum f. sp. cubense (Foc), agente causal do mal-do-Panamá da bananeira. Para tanto, discos de rizoma foram tratados com 200 µL de suspensão dos antagonistas (109 UFC mL-¹) e inoculados com patógeno. O material foi disposto em caixas plásticas tipo gerbox e incubado em BOD a 25 ºC, por doze dias. Após, a área dos discos de rizoma (mm²) colonizada por Foc dos discos foi quantificada por meio de imagens digitais. O ensaio foi montado em delineamento inteiramente casualizado, com quatro repetições e três discos por repetição. Discos não tratados e inoculados compuseram o controle. Com base nos resultados obtidos, foi possível verificar a eficiência do método na seleção de agente de controle biológico, uma vez que foi possível o agrupamento dos 26 isolados utilizados em cinco clústeres distintos, sendo que isolados pertencentes a quatro destes grupos (do '1' a '4') foram capazes de proporcionar a redução da colonização por Foc. Os isolados com maior potencial de inibição foram520EB, 993EB e 531EB com 343,3, 344,1, 364,8 mm2, respectivamente, promovendo inibição variando de 53 a 56 % da área colonizada quando comparados com ao controle (782,6 mm²).

An Improved Phenotyping Protocol for Panama Disease in Banana.

Fusarium oxysporum (Fo) belongs to a group of soil-borne hyphomycetes that are taxonomically collated in the Fusarium oxysporum Species Complex (FOSC). Hitherto, those infecting bananas were placed in the forma specialis cubense (Foc). Recently, however, these genetically different Foc lineages were recognized as new Fusarium spp. placed in the Fusarium of Banana Complex (FOBC). A member of this complex F. odoratissimum II-5 that uniquely comprises the so-called Tropical Race 4 (TR4), is a major problem sweeping through production zones of Cavendish banana in several regions of the world. Because of this, there is an urgent need for a phenotyping method that allows the screening for resistance to TR4 of large numbers of banana genotypes. Most Fusarium species produce three types of spores: macroconidia, microconidia and the persistent chlamydospores that can contaminate soils for many years. Inoculum production has been an important bottleneck for efficient phenotyping due to the low or variable number of conidia and the elaborate laboratory procedures requiring specific infrastructure. Here, we report a rapid, simple and high-yielding spore production method for nine F. oxysporum formae speciales as well as the biocontrol species Fo47 and Fo618-12. For Fusarium spp. causing Fusarium wilt or Panama disease of banana, we used the protocol for four species comprising the recognized physiological races, including Tropical Race 4 (TR4). We subsequently tested the produced inoculum in comparative inoculation trials on banana plants to evaluate their efficiency. All assays resulted in typical symptoms within 10 weeks; significant differences in final disease ratings were observed, depending on inoculum concentration. Pouring inoculum directly onto banana plants showed the most consistent and reproducible results, as expressed in external wilting, internal discoloration and determined by real-time PCR assays on entire rhizomes. Moreover, this method allows the inoculation of 250 plants per hour by one individual thereby facilitating the phenotyping of large mutant and breeding populations.

Fusarium Wilt of Banana: Current Knowledge on Epidemiology and Research Needs Toward Sustainable Disease Management.

Banana production is seriously threatened by Fusarium wilt (FW), a disease caused by the soil-borne fungus Fusarium oxysporum f. sp. cubense (Foc). In the mid-twentieth century FW, also known as "Panama disease", wiped out the Gros Michel banana industry in Central America. The devastation caused by Foc race 1 was mitigated by a shift to resistant Cavendish cultivars, which are currently the source of 99% of banana exports. However, a new strain of Foc, the tropical race 4 (TR4), attacks Cavendish clones and a diverse range of other banana varieties. Foc TR4 has been restricted to East and parts of Southeast Asia for more than 20 years, but since 2010 the disease has spread westward into five additional countries in Southeast and South Asia (Vietnam, Laos, Myanmar, India, and Pakistan) and at the transcontinental level into the Middle East (Oman, Jordan, Lebanon, and Israel) and Africa (Mozambique). The spread of Foc TR4 is of great concern due to the limited knowledge about key aspects of disease epidemiology and the lack of effective management models, including resistant varieties and soil management approaches. In this review we summarize the current knowledge on the epidemiology of FW of banana, highlighting knowledge gaps in pathogen survival and dispersal, factors driving disease intensity, soil and plant microbiome and the dynamics of the disease. Comparisons with FW in other crops were also made to indicate possible differences and commonalities. Our current understanding of the role of main biotic and abiotic factors on disease intensity is reviewed, highlighting research needs and futures directions. Finally, a set of practices and their impact on disease intensity are discussed and proposed as an integrative management approach that could eventually be used by a range of users, including plant protection organizations, researchers, extension workers and growers.