EMERGENCE AND EPIDEMICS OF A NEW DISEASE IN ARAGUAIA VALLEY: ANTHRACNOSIS OF COTTON CAUSED BY Colletotrichum truncatum, VARIETAL REACTION AND DIFFICULTIES OF CHEMICAL CONTROL EPIDEMIAS DA ANTRACNOSE DO ALGODOEIRO NO VALE DO ARAGUAIA CAUSADA POR Colletotrichum truncatum REAÇÃO DE CULTIVARES E DIFICULDADES DE CONTROLE QUÍMICO

Cotton culture (Gossypium hirsutum L.) is trending in an upward expansion amidst Brazilian “cerrado”. Due this growth, new pathosystems are growing in incidence on tropical fields in the region of Araguaia Valley MT. Surveys and material collection were conducted out in production areas on two counties (Bom Jesus MT and Canarana – MT), both regions represents a total amount of 50.000 hectares of cultivated area. The region also is characterized by succession areas previously sowed with soybean plants (main cover crop season). Previously surveys revealed the incidence of target spot (Corynespora cassiicola), ramularia spot (Ramularia areola) and cercosporiosis (Cercospora gossypii). Disease incidence is commonly observed on several crop management methods and cultivars. Due to a reduction in efficacy of chemichal control by fungicides spray programs, this disease is increasing and spreading in a faster rate in production areas with previous harvested soybean at the biggest soybean producer group in the world (Bom Futuro). Plants on field were selected due to differences in symptoms, and isolation methods were carried out on PDA (potatodextrose-agar) before “in vitro” pathogenicity tests conducted on seedlings, detached leaves and bolls (growth chamber conditions of 23C / photoperiod of 12 hours). After Colletotrichum dextructor sp. pathogenicity confirmation, bioassays were carried out with several different fungicide’s groups (i.e. registered for usage on Brazilian jurisdiction). This test consisted of the employment of a solution with 500 ppm of each different fungicide/a.i. that is applied on infected bolls and detached leaves who were inoculated with the target pathogen (concentration of 10 conidia per mL). This test was followed by an infection (%) evaluation during 10 days of incubation. The incidence/prevalence index (%) was also evaluated at different parts of the plant (lower, middle, and upper canopy) on different cultivars/genotypes (130-150 days after sowing). A standard level of control by different fungicides, ranged between 0 to 100% of control. Two groups of cultivars/genotypes were separated after differences on resistance response, one with susceptible traits (FM 985 GLTP) and other with partial resistance traits. No immunity response was observed. We suggest that new efficacy tests should be carried out with combination of varietal response (resistance or tolerance) combined with other chemical fungicides for better understanding of synergism or positive interaction. Valuable information will highlight the best association for greater varietal response and yield against this necrotrophic pathogen (higher B0 – initial inoculum) survival during successive years of rotation main crop (cotton x soybean). Isolated spray of benzimidazoles, cupric (except cuprous oxide) and triple associations with triazoles or triazolinthione (prothioconazol) combined with strobilurins and carboxamides should be wisely administrated to manage this disease due to low efficiency (below 50%). New field and laboratory essay must be carried out to input data about resistance risk and clarify damage levels on leaves and bolls impacting yield.


INTRODUCTION
Cotton plants (Gossypium hirsutum L.) usually are infected with fungal, bacterial, viral, and nematode generating depreciation of plant growth that will affect boll yield and quality of fibers. Foliar epidemics of fungi such as Ramularia areola, Cercospora gossypii, Alternaria macrospora, Stemphylium solani, Rhizoctonia solani and Colletotrichum gossypii var. cephalosporioides have JULIATTI, F. C. et al Biosci. J., Uberlândia, v. 36, n. 6, p. 2120-2132, Nov./Dec. 2020 http://dx.doi.org/10.14393/BJ-v36n6a2020-56826 been reported in Brazil and are growing in importance in recent years. The damage by an aggressor is fungus (biotrophic or necrotrophic), or bacteria in vegetative or reproductive parts, can reflect in addition losses to industrial product (cloth) performance. Adding up the Covid-19 Pandemic, after the oil prices fell worldwide contributed to the stimulation of synthetic fiber production that ended up affect the textile industry cost production per pound of cotton fiber weight. Ramularia spot is the main leaf disease, the optimum conditions for its development and development of field epidemics are stated in literature to a combination of 20-28ºC and leaf wetness of at least 6 hours daily (mild rains or foliar dew). Also, incidence of sun (photo stimulation) on leaves, can lead to an abundant sporulation of the pathogen leading to defoliation of plants who also impairs the development of cotton bolls. Additional incidence of other leaf diseases caused by Cercospora spp., Alternaria spp., Colletotrichum gossypii var. cephalosporioides (ramulosis) could extend several impacts on plant overgrowth affecting the final products. Colletotrichum gossypii var. cephalosporioides characteristic conidia are ovoid with size of 6 to 17 µm with hyaline conidiophores generating numerous acervuli spikes, authors suggested that higher incidence usually is related to long periods of prolonged rain and its teleomorph form Glomerella gossypii Edg. was also described in Venezuela (COSTA, FRAGA, 1937;MALAGUTI, 1955). The pathogenicity and inoculation of several isolates can be confirmed by relatively simple methods, like an application of a solution of water and of conidia spray on younger cotton plants.
In the last 4 years, cotton became a great cover crop ("safrinha") option to Brazilian farmers after succession/rotation with soybean in previously first option of harvest season ("safra"), this led to a certain difficulty in obtaining superior genotypes with broad resistance to disease complex that affects both crops (surplus in initial inoculum B0) . Genotypes such as Redenção and IAC 20 presents higher levels of resistance to leaf disease complex those as well are being unused over the years and became obsolete sources after showing lower yield on field (POLIZEL, JULIATTI, HAMAWAKI, PENNA, 2008). Currently, the most planted cultivars/genotypes in Brazil are the cultivar FM 985 GLTP and FM 911 GLTP, both shows superior boll quality and yield on the Araguaia Valley (northern region Mato Grosso state), but showed as well higher susceptibility that grew in importance in the last season (unknow etiology). This new disease was observed by farmers and growers in the region of Bom Jesus -MT and Canarana MT, they also reported that several fungicides spray (9-10 applications) could not reduce the higher levels of incidence thus affecting fiber quality and yield (kg.ha -1 ). Several fungicide programs have been used by farmers, including the intense spray and rotation of several (multi-sites, strobilurins, triazoles and carboxamides) active ingredients. This also could be main reason of a growing concern that could be related to fungi isolates selection process on field after a wrong choice in timing spray and application interval in combination of fungicides groups without research data to support the Integrated plant disease management (IDM).
Vale do Araguaia -MT is a production region inside Brazilian transition between savannah (cerrado) and Amazon rainforest (equatorial climate), this region is characterized by well distributed precipitation closely to 2.000 mm by year during wet season (7 months) and a prolonged dry period (another 5 months). Cotton became a popular choice for growers after higher performance of yield and quality of cotton plants, who generates fibers with desirable characteristics. The averages in temperatures usually ranges between 25 to 28 °C . Leaf wetness after prolonged dew periods and optimum temperature contributes to the development of several foliar fungal etiologies. In this scenario, of a chemical control losing efficacy compromising yield after the vegetative development and fiber loss quality being affected by growth regulators promotion, there is an urgent need to carry out systematic and periodic surveys on the main diseases that occur in this culture that is strategic for agribusiness in Brazil and Araguaia region. The development of data to support sustainable management programs with fungicides (chemical and biological combination) in different cultivars/genotypes must be priority to cotton phytopathological research.

Location of collection and sampling experiments
The collections of symptomatic material were carried out in Bom Jesus -MT (Fazenda Malú of the Bom Futuro group -MT) and Canarana -MT (Fazenda Cocal -Bom Futuro group -MT). The collections were carried out in areas with higher incidence and severity of phytopathogens in the lower, medium, and upper cannopy of cotton plants in all cultivars/genotypes used experimentally and commercially by the farmer group. Samples of leaves, bolls and stems were stored in plastic bags and placed on foam boxes with ice (10 to 15 ºC) and JULIATTI, F.

Isolation in culture medium from possible plant pathogens
To perform direct fungi isolation, sections of infected tissue were marginally cut close to the lesions (peripheral) or sporulation structures and placed in a moist chamber on Gerbox (ZAMBOLIM, PEREIRA, 2012), to obtain pure culture in PDA culture medium, routinely used to obtain isolates. Candidate cultures without the presence of saprophytic fungi, were selected to represents isolates with potential for association with the new pathology (disease). The candidate cultures obtained were grown in test tubes with PDA and stored by suitable methods for pathogenicity preservation. At least one hundred different samples were identified with presence of viruses or bacteria that could be contamination sources and were discarded as well (JULIATTI and POLIZEL, 2003;WATHINS 1968 and KIRKPATRICK, T.L.; ROTHROCK 2001).

Inoculations and pathogenicity tests
Inoculations were carried out on detached leaves and green cotton bolls not yet fully ripen or in the process of burst. Leaves and cotton bolls were superficially disinfected with a combination of solutions with 50 % alcohol after submitted to in 2 % sodium hypochlorite, and then washed with sterile water. Samples (separating bolls from leaves) were packed in Gerboxes with a thin layer of moisted sterile germitest paper and then inoculated with conidia at a concentration of 10 4 per mL. Incubations were performed in growth chambers (GC) chambers at 12-hour photoperiod in 23 °C for 10 days. The evaluations consisted in incidence data whether the sample had pathogenic infection. Pair of leaves (unifoliolate leaf) of seedlings (cultivar NuOpal) were also inoculated, to confirm the etiology of the fungi involved (Koch postulate).

Reaction of Genotypes / cultivars
Genotypes resistance ranking was carried out in field with visual assessment of the prevalence index in each cultivar/genotype present in Bom Futuro group farms (50.000 ha), located in the municipalities of Canarana -MT and Bom Jesus -MT. Commercial plots of the following were evaluated: Fibermax ( . Sampling was carried out in the three parts of the plant (lower, medium and upper canopy) to determine the prevalence index in each cultivar/genotype canopy region. The average severity was also assessed based on the percentage of disease area on leaves at each production plot on field (stand) ranging from 0 to 100 % area of infection. All field plots previously reported were sprayed at least 8 times. Healthy bolls and leaves were harvest in two cultivars (DP 1746 and FM 985 GLTP), both respectively corresponded to a collection on a field experimental area inside the research station JuliAgro B, G & P and LAMIP -UFU (growth chamber) both places are located in Uberlândia -MG. This biological material was treated for asepsis in solutions containing alcohol, sodium hypochlorite and sterile distilled water. Samples them were packed in Gerbox containing sterile Germtest paper for incubation after spray with 55 different commercial fungicides (Table 1), at doses of 500 mg. Kg -1 (ppm). These products consisted in registered or in process of crop registration (RET 3 -Temporary registration Level 3 -MAPA). The fungicides evaluated can be an important strategy in the management of this new disease. The fungicide spray was carried out by a hand manual sprayer used in gardening. The material was sprayed at run off point (detached leaves and cotton bolls) and them immediately a conidial suspension containing Colletotrichum truncatum conidia at a concentration of 10 4 conidia per mL was inoculated. Incubation took place at 23ºC for 10 days. Two repetitions were performed over time (separated by 10 days) the overall experiment was reproduced another time. After incubation, the percentage of severity (%) of diseased boll and leaf area was determined. The experimental design was completely randomized (CRD).

Seedling inoculation
Cotton seedlings with younger leaves were sprayed with all the fungicides reported in Table 1 (500 mg. Kg -1 ) and then inoculated with the virulent isolate (Colletotrichum dematium) obtained in production area of Canarana -MT at a concentration of 10 4 conidia per mL. Incubation was carried in a growth chamber for 15 days. At least a 24-hour period inside a moist chamber (100 % of humidity) is needed for the germination and infection of conidial initial inoculum (B0) on leaves and plant stem. Fifteen days after inoculation, the seedlings were evaluated by percentage of severity (%). The experiment was carried twice replication over time and overall reproduction (vessels). Plots consisted of a single cotton plant grown up in 200 mL plastic cups, containing organic substrate.

Data analysis
The data were analysed using the SASM-AGRI software (ALTHAUS, CANTERI, GIGLIOTI 2001;CANTERI et al. 2001). The averages were grouped in the same software by the Scott and Knott test at 1% probability (p <0.01) (CANTERI et. al. 2001).

Analysis of infected material from Canarana MT and Bom Jesus MT
After sampling and processing infected leaves and cotton bolls on GC (growth chamber), the following fungi were isolated in culture media: Colletotrichum spp., Alternaria alternata and Fusarium spp. The fungi with falcate conidia with dimensions ranging from 3 to 5 microns (width) and 25-30 microns (length) were selected in visual inspection on microscope (40 x). The forms of conidia's visualized were similar the Colletotrichum truncatum from soybeans, since this pathogen is not reported in cotton plants the name was attributed to the newly pathogen. Further studies in molecular biology must be conducted for better understanding and discard the hypothesis that the Colletotrichum truncatum how adapted to a new host. This fungus is prevalent in infected leaves, stem, and cotton bolls (figures 1, 2, 3 and 4).

Pathogenicity Test
The pathogenicity test after inoculation of the fungus Colletotrichum truncatum was confirmed, both in leaves and in detached cotton bolls. The phytopathogen grows and sporulates in PDA medium easily, at a temperature of 23 ºC and photoperiod of 12 hours (figures 4 and 5).

Evaluation of fungicide efficiency in detached leaves and bolls
Fungicides (active ingredients -a.i.), trade names, chemical groups used in the essay are presented in Table 1, some are being used to supress JULIATTI, F. C. et al Biosci. J., Uberlândia, v. 36, n. 6, p. 2120-2132, Nov./Dec. 2020 http://dx.doi.org/10.14393/BJ-v36n6a2020-56826 disease development, but without field data to better clarify and solve the hypothesis of a defined strategy for this phytopathogen evolution on cotton cultivation in Vale do Araguaia -MT. All fungicides were adoppted at a concentration of 500 ppm (mg.Kg -1 ). If we trace a limit in consideration of evaluated fungicides (Table 1) with a standard control percentage above 80%, 28 fungicides with possibilities of use in the management of the disease were categorized for foliar and 33 fungicides for cotton boll spray. We also need to establish a clear strategy for rotation of fungicides since some fungicides showed bellow of 10% control for both leaf and boll infection (reproductive and vegetative stage). The fungicides that showed 100 % control in this initial screening for leaves and cotton bolls were duo and trio associations of protioconazole + mancozeb, cyproconazole, difeconazole + cyproconazole, mancozeb, procimidone, tebuconazole and azoxystrobin + mancozeb + cyproconazole. The inefficacy of carbendazim and fungicides derived from metallic copper (copper oxychloride and cuprous oxide) stands out. Proticonazole + pydiflumetofen, fluazinan + methyl thiophanate, metiran + pyraclostrobin, tetraconazole + azoxystrobin, chlorotalonil + tebuconazole, fentin hydroxide, trifloxystrobin + tebuconazole, protioconazole, trifloxystrobin, chlorotoxin, chlorotoxin, chlorothalonil, ciproconazole, sulphur SC, azoxystrobin + mancozeb, mancozebe, fenpropimorph and picoxystrobin + benzovindiflupyr showed a minimum control efficacy around 70 % for leaf and boll infection. We also noted that fungicides with the lowest control efficacy were biological, cupric, benzimidazoles + triazoles + strobilurines, benzimidazoles, carboxamide + strobilurines, carboxamide + strobilurines and carboxamide + strobilurine + triazolintione which presented a control percentage below 50 % ( Table 2) for both vegetative and reproductive organs. This type of information is very important to establish integrated management systems, or rotation of fungicides or active ingredients when seeking the rational management of the new epidemic focusing on biological control assisting chemical.

Reaction of genotypes by prevalence index in the upper, middle and lower thirds of plants under field conditions
The Table 3 shows the cultivars or genotypes cultivated in the Araguaia Valley or during the experiment period and their reaction to anthracnose considering the prevalence index in the plant in the lower, middle and upper canopy of the plant, as well as the severity of symptoms and the consequent leaf destruction by Colletotrichum truncatum. The cultivar with the highest levels of severity was FM 985 GLTP, with an average severity in the evaluation on field up to 70 %, followed by FM 975 WS with 30 % severity (prevalence index). The superior severity in the cultivar FM 985 GLTP could be hypothesized of its representative growth area, since it's one of the most cultivated cultivar/genotypes in Araguaia Valley -MT and given its productivity and fiber characteristics (source of cellulose, sugar and proteins). In some locations, the cultivar even showed a reduction of more than 60% in productivity. Given the higher virulence of this disease, its estimated the yield losses in cotton areas during the last two seasons could have reached more than 3.000 kilograms of cotton bolls per hectare. CONCLUSIONS A level of control by different fungicides ranging from 0 to 100% was confirmed for leaves and bolls.
The prevalence index (%) was also evaluated, considering the three regions of the plant (lower, middle and upper canopy), on different genotypes under field conditions in the final stage of plant production (130-150 days).
The genotypes that were considered in this initial screening as susceptible was FM 985 GLTP and others were gauged as with presence of some certain degree of partial resistance.
No genotype was immune to the pathogen. It is suggested that programs and management with fungicides be revised in order to find the best active ingredients for this new pathogen, Also new studies with fungicides (biological chemical) being associated with genotypes with greater varietal resistance should be conducted for this necrotrophic and destructive pathogen surviving phase (considering lowering the initial inoculum B0).