Sanitary analysis , transmissibility and pathogenicity of fungi associated with cashew nuts

There are few reports of transmission of fungus associated with cashew nuts (Anacardium occidentale L.) in Brazil and worldwide. Thus, the aim of this study was to evaluate the incidence and severity of anthracnose on leaves and fruits (nuts) of cashew, nuts sanitary analysis, nuts-seedling transmission and pathogenicity of fungi associated with cashew nuts. To this end, leaves and nuts of cashew collected in the cities of Gurupi and Formoso do Araguaia , Tocantins state, Brazil, were used. The anthracnose average incidence and disease index were evaluated by observing the typical symptoms of the disease. The nuts were subjected to sanity analysis, by the filter paper method and transmission test via nuts for the seedlings. High anthracnose incidence and severity were observed in leaves and cashew s nuts. Aspergillus sp. Cladosporium sp. and Colletotrichum gloeosporioides showed high incidence in all nuts samples analyzed. There was high anthracnose transmission by the nuts for the seedlings. Only C. gloeosporioides was pathogenic to cashew seedlings. Higher germination rates were observed with lower levels of lesions on the external surface. The fungal association with cashews nuts damaged generally normal seedlings development and quality.


INTRODUCTION
The cashew (Anacardium occidentale L.) is a tropical native species from Brazil spreading almost all-Brazilian territory (Coutinho et al., 2016).Constitutes an income source for the population and has great economic and *Corresponding author.E-mail: gilrsan@uft.edu.br.
Author(s) agree that this article remain permanently open access under the terms of the Creativ e Commons Attribution License 4.0 International License social importance, especially in the Northeastern region.Although most of the cashew production is located in the country coastal region, a large increase in production area is occurring in Semiarid and Cerrados (Brazillian savanna) regions, probably due to favorable conditions for a higher fruits (nuts) and pseudo fruits (flower peduncle) quality than in any ot her c ount ry region.The cashew propagation is carried out through nuts (seeds) or vegetative parts of plant, the latter being the most recommended, as it ensures obtaining more uniform fields with desirable characteristics and more productive (Dendena and Corsi, 2014).The planting through nuts is still widely used in several regions.However, the use of contaminated and/or infected nuts by pathogens can result in lower plant population and lower production and pseudo fruits quality because they are vehicles of many pathogens that serve as initial inoculum and can cause epidemics in the orchard.
In most plants, it is mainly transmitted t hrough seeds, but also through plant infected parts.Symptoms may appear on leaves, fruits and seedlings, occurring in all plant development stages and can cause seedlings necrosis and death (Dendena and Corsi, 2014).The fungus may attack during all the months in the year, but cause great er damaged in the rainy season and when issuing news hoots.The diseas e management was carried out using sanitary practices and chemical control (Figueirêdo et al., 2012).The use of resistant clones is also should be adopted, however, all commercially available clones are susceptible to disease (Figueirêdo et al., 2012).
Healt hy seed must have a high germination rate and be free of seed-borne pathogens and healthy seeds are recognized as an import ant input in any agricultural production system.However, there is a little information available about the transmission of fungi through the cashew nuts in Brazil and worldwide, which makes this study of great importance.Based on the expos ed, the aim of this study was to evaluate the incidenc e and severity of anthracnose on leaves and nuts of cashew nuts, sanitary analyzes, cashew nuts -seedlings transmission and pathogenicity of fungi associated with cashew nuts.

Sam ple collection
The study w as carried out from August, 2013 to October, 2014.An amount of leaves and nuts w ere collected from a total of 20 cashew trees, previously selected, from tw o cities of Norther n region Brazil, Gurupi (11° 43' S; 49° 04' S) and Formoso do Araguaia (11° 47'45''S; 49° 31'52''W), State of Tocantins.After the collection, the nuts w ere mixed for performing the tests.

Pathogen identification
Cashew leaves fragments w ith anthracnose sy mptoms w ere subjected to sterilization by immersion in 50% alcohol solution for 30 s, 1% sodium hypochlorite for 40 s and three w ashes in sterile distilled w ater.After, the leaf fragments w ere placed on Petr i dishes containing potato dextrose agar ( PDA) culture medium, and maintained in a grow th chamber at 25°C ± 2°C for seven days.After incubation, the fungi identification w as carried out by analyzing the fungal structures, w ith the aid of an light microscope.The observed structures w ere compared w ith structures described in the literature (Barnett and Hunter, 1972).
The identified Colletotrichum sp.isolates w ere grow n on PDA medium and incubated for seven days for further molecular identification at spec ie level.For DNA extraction, discs containing the mycelia w ere excised and inoculated into 60 mL of liquid medium Yeast extract (10 g yeast extract, 10 g dextrose, 1 L distilled w ater) w ith antibiotic ( Clor anfenicol -200mg/L).For mycelial grow th, they w ere kept under agitation (120 g) for seven days at 27 °C.The mycelium w as separated from the liquid medium by vacuum filtration.After w ashing w ith sterile distilled w ater the harvested mycelium w as ground to pow der in liquid nitrogen, w ith a pestle and a mortar.DNA w as extracted from the pow dered mycelium us ing a CTAB method adapted from Zolan and Pukilla (1986).Briefly, about 0.5 g of this pow der mycelium w as then transferred to a micro centrifuge tube, and 1 mL of preheated (65ºC) extraction buffer (2% p/v de CTAB; 2.5% p/v de PV P; 2M de NaCl; 100 mM Tris-HCl (pH 8.0), 25mM EDTA (pH 8.0), 2% (v/v) ßmercaptoetanol) w ere added.The pow der w as mixed w ith the buffer, vortexed briefly, and stor ed at 60°C for 40 min and then centrifugate at 11,000 g for 10 min.After separation of the tissue from the aqueous phase, an equal volume of chloroform/isoamy l alcohol (24:1) w as added.The RNA w as degraded by treatment w ith RNase A (50 mg/mL) for 30 min at 37°C.The DNA w as precipitated by adding 2.5 volumes of absolute ethanol and pelleted by centrifugation for 15 min at 12,000 g.The pellet w as washed w ith 70% ethanol and resuspended in ultrapure w ater.The DNA concentration and pur ity w ere measured using a spectrophotometer.

Incidence and anthracnose severity
Fifty cashews leaves and nuts w ere randomly obtained from 16 cashew s tree to quantify the anthracnose incidence and severity.A completely randomized experimental design w as used w ith 11 replicates.Average incidence of anthracnose on leaves and nuts was expressed in percentage us ing: % incidence = ∑ . Where x = number of infected leaves or fruits and n = total number of leaves or fruits observed.In the assess ment of anthracnose average severity on leaves and nuts, w e used rating scale 0-4 adapted from Santos et al. (2005), w here 0 = no symptoms; 1 = sy mptoms at 10% of the affected area; 2 = 11 to 25% of the affected area; 3 = 26 to 50% of the affected area; and 4 = more than 50% of the af fected area.Later, the disease index ( DI) was determined using the McKinney index formula (1923) as show n: DI= ∑ .Where: ΣF( n) = the sum of notes frequency; TNL= total number of examined leaves; e MN = maximum scale note.

Sanitary analysis of cashew nuts
For this assay, the blotter test method w as used, follow ing the description in the Rules for Seed Testing -RAS (Bras il, 2009).The experimental design w as completely randomized, w ith 16 replications.Initially, the nuts w ere w ashed in w ater and subjected to sterilization in 1% hypochlorite, 70% ethanol and distilled w ater.The nuts w ere placed in a transpar ent box (gerbox), w ith 25 nuts on each box, on tw o layers of germitest paper previous ly autoclaved and moistened w ith sterile w ater.The boxes containing the nuts were kept in incubation chamber at 25°C ± 2 °C for seven days, w ith alternate light.After incubation, the fungi identification w as carried out on the nuts as described above.The values of results w ere expressed in percentage of incidence of each fungus.When it w as not possible the fungus genera c lassification using the structures present, the hyphae of these fungi w ere collected and placed on PDA medium, for later identification.For confir mation of Koch's principles, the fungi found in the nuts w ere isolated on PDA medium.To test the pathogenicity of these fungi to cashew plants, the conidia w ere inoculated by spraying at a concentration of 1 × 10 5 conidia mL using a manual sprayer in cashew seedlings at 40 days after emergence.Then the seedlings are stored for 24 h in a moist chamber until disease symptoms onset.

Cashew nutseedling transm ission
For the cashew nutseedling trans mission assay, 50 nuts in three replications, totalizing 150 cashew nuts w ere sow ed w ithout disinfestations, on plastic trays containing autoclaved sand.After the sow ing, the trays w ere transferred to a greenhouse w ith temperature of 30°C ± 5°C.Daily irrigation w as performed w ith the aid of a w atering pot.The seedlings evaluation w as made 15 days after the emergence by observation of the character istic sy mptoms.The fungi trans mission w as assessed by considering the percentage of seedlings w ith anthracnose sy mptoms on leaves, steam and cotyledon.It is also evaluated the percentage of ger minated and non-ger minated nuts and the percentage of dead seedlings.To identify the fungi spec ies in the seedlings lesions, the injured fragments of the primary root, stem, cotyledon and leaf w ere removed and submitted to the isolation process as described already.

Effect of anthracnose severity on the cashew nuts in the germ ination
The nuts w ere selection based on external surface visual observations and separated according to damage level using a rating scale adapted from Santos et al. (2005).We used a completely randomized design w ith three replicates; the treatments consisted of five different severity levels and each replicate w as constituted by 50 cashew nuts.The sow ing w as made on polypropy lene trays, w ith autoclaved sand as substrate.After sow ing, the substrate w as moistened and the trays w ere kept in a greenhouse w ith temp erature of 30°C ± 5°C.The assessment w as carried out over 30 to 50 days after the seedlings emergence.It's considered as ger minated nuts, those that gave rise to nor mal seedlings w ith all the essential structures (Bras il, 2009).The trans mission w as observed in nor mal and not nor mal seedlings by count seedling w ith lesions on leaves.The pathogen identification w as carried out as described above.

Statistical analysis
The data of anthracnose averange incidence, disease index and the relationship betw een infection severity and anthracnose trans mission w ere subjected to analysis of variance (A NOVA) and differences betw een means w ere deter mined according to Tukey's test, at 5% probability.The data expr essed as percentage w ere transformed into arcsin x/100 for statistical purposes, w hen necessary.The graphics w ere plotted using the Sigma Plot (version 10.0) softw are and the statistical procedures w ere held w ith Sisvar (version 5.3) softw are.

Pathogen identification
All isolates from cashew leaves with anthracnose symptoms were identified, based on morphological aspects, such as conidial morphology and presence or absence of setae, with similar characteristics to those described for species belong to the genera Colletotrichum sp.However, because definitive identification of Collet otrichum species based on morphology is difficult due to overlapping ranges of conidial and colony characteristics and the fact that variation in morphology is accepted for isolates within species, a number of molecular met hods have been us ed to characterize species of Colletotrichum (Uaciquete et al., 2013).
Based on the analysis of laccase gene with the s pecific primers, this study was able to confirm that the cashew isolate were identified as C. gloeosporioides.The primer tested in our study generated amplification products of 505 bp (Figure 1) corresponding to C. gloeosporioides laccase gene described by Shi et al. (2008).Morphological and molecular characteristics performed in this study confirmed C. gloeosporioides as the causal agent of cashew anthracnose.These finds corroborate with the described by and Uaciquete et al. (2013) that C. gloeosporioides is commonly reported as the causal agent of anthracnose in cashew trees.The identification and confirmation of the Colletot richum species pathogenic to cashews is crucial to t he development of more efficient control strategies, besides contributing for a better understanding of the anthracnose disease epidemiology.

Incidence and anthracnose severity
There was a high anthracnose incidence on the leaves (68.1% ) in this study (Figure 2A).The ant hracnose incidence on the leaves differed statistically (Tukey, P≤0.05) to the anthracnose incidence in nuts (31.1%).Distinct of the observed in anthracnose incidence, disease index was higher in the nuts than in the leaves, 34.7% and 31%, respectively (Figure 2B).Anthracnose of cashew is a disease known in several regions where it grows cashew, being its incidence always related to the region climatic conditions.The climatic conditions of the Brazilian Northern region are favorable to the growth and proper development of cashew tree.However, high temperatures and high relative humidity (around 85%) found here provide better pathogen development and increasing severity on leaves, stems and fruits, as observed in these study.Long-t erm ex posure to disease promoting or predisposing factors can increase disease development in perennial hosts (Frare et al., 2016), as cashews trees.Host nutritional status is also an important abiotic factor that may be related to increased disease in orchards without proper maintenance, such as where the study was conducted.

Transport of fungi associated with cashew nuts
The results showed a high incidence of Aspergillus sp.(49.71%) in all tested samples (Figure 3).The incidence of the genus Cladosporium sp.(17.14% ), Penicillium sp.(11.42%),Pestalotia sp.(0.5%) and C. gloeosporioides (26.85%) were also observed.Among all fungi found in the cashew nuts only C. gloeosporioides was pathogenic to cashew seedlings.
The fungi genus Aspergillus, Cladosporium and Penicillium found in large quantities in the assessed cashew nuts are involved in the decay process of the nuts at sowing stage, by interfering directly in germination rate and seed vigor.These fungi may attack different kinds of seeds, once they are able to survive on several  substrates, and are resistant to a variety of environmental conditions (S harma and Kulshrestha, 2015).The high incidence of these fungi makes necessary the use of some control method, since it affects directly the final product quality, particularly cashew nuts intended for human feeding.It must be alert to contaminants fungi, Aspergillus sp. and P enicillium sp., which always occurs in higher percentages, besides being potentially mycotoxin-producing.Thus, they pose a potential hazard to consumers' health.The presence of some metabolites and traces of aflatoxin in cashew kernels has been demonstrated (Milhome et al., 2014).It is estimated that about 10% of the annual production of cashew nuts is unsuitable kernels for industrial processing and for  human feeding, due to the fungi pres ence, mostly (Adeigbe et al., 2015).Despite the well-known antibiotic properties of the cashew nut testa and shell (Schulz e-Kaysers et al., 2015), a wide spectrum of fungi can survive saprophytically on cashew seeds.Following seed germination, young tissues may be susceptible to some of these fungi.The attack caused by fungi present in nuts on the cotyledons, causes seedling nutrients loss and healthy tissues destruction, resulting in a not normal seedlings development, with wrinkled and small size leaves and twisted stems.
The fungus C. gloeosporioides is commonly report ed as causing rot in nuts (Sarkar, 2016), being found in t he present study with an incidenc e of over 50% in the evaluated samples.However, the infection seeds not ensure the pat hogen transmission, because in addition to the host, the factors linked to the environment and the pathogen must be considered.The fungus can survive in dead tissue and in the soil and can be spread by rain splash, wind, insects, etc. Nonet heless, when t he pathogens find healthy tissue it can cause severe injury in favorable environmental conditions, and greatly decrease of nuts and peduncle production ( Dendena and Corsi, 2014).

Cashew nutseedling transmission
There are several factors limiting the efficiency of pathogen transmission by seeds.Each pathogen has specific requirements of temperature, humidity, oxygen, quantity of nut rients, mechanical injury, seed age and the presence of antagonistic microorganisms on the seed (Rego et al., 2012).In this study, anthracnose transmission of cashew nuts for the seedlings was verified through the percentage of seedlings with disease symptoms (Figure 4).In cotyledons was observed anthracnose incidence on 39% of seedlings and leaves symptoms were observed in 41% of the seedlings.For stem symptoms, there were few plants with ant hracnose symptoms (8%).A similar result was found by Lopez and Lucas (2002) under laboratory conditions, they noted that 33% of seedlings emerging from non -treated cashew seeds had nec rotic lesions on cotyledons, hypocotyls or epicotyl.C. gloesporiodides was detected in red pepper (Capsicum annuum L.) seeds and is associated with anthracnose in this crop.This pathogen can be transmitted from the endos perm tissue for hypoc otyl and rootlets and is located generally on the surfac e of seeds, in the endosperm and embryo (Ali et al., 2016 ).The percentage of germinated nuts, with and without disease symptoms it was low (57%) and the quantity of nuts which do not germinate was 24%, while the number of dead seedlings was 9% (Figure 4).Seeds pathogen carriers may result in failures germinati on, seedling death or development of low-quality plants, thereby compromising production (Coutinho et al., 2016), as observed in this study.

Effect of anthracnose severity on the ca shew nuts in the germination
There was a higher germination rates in nuts with lower lesions levels on the outside surface (Table 1).The nuts germination started at 19 days after sowing; however, it was found that there was a delay in some nuts and uneven germination.Nuts with a high degree of severity (note 3 and 4) gave greater number of plants with leaf spot and anthracnose symptoms.In a humid chamber, the presence of C. gloeosporioides in the injuries was found.The transmission in this case may be conditioned to the severity of the seed fungal infection, or be relat ed to the lesion size and the amount present in the seed inoculum.Silva et al., (2013) using selected seeds with different levels of C. lindemuthianum incidence observed variation from 70 to 100% in the transmission rate of seeds with incidence of 1 to 5% the pathogen.Germination, while above 50%, demonstrat es the damaging effect of the fungi on the vigor of the nuts and possibly the onset of leaf spots.In nuts with higher levels of severity were obs erved higher rates of not normal seedlings, beside the highest number of nuts that did not complete germination, and seedlings that died aft er emerge.These effects may be related to the fact that most affected nuts having lower reserve level to assist the early seedling development, besides the high incidence of fungi that probably causes more damage to seedlings.It was also found that despite of the good external appearance in some nuts, not always indicate the absence of pathogens, since many of them may be in a latent state waiting for adequate moisture conditions for manifesting.
The diffic ulty of obtaining healt hy seedlings, due to the number of pathogens that are associated with cashew nuts, might be one of the causes of low germination and reduced development of seedlings coming from seeds.To Marques et al., (2013) the dwarf cashews seed germination begins on the 10th day aft er sowing and continues until the 25th day, however normally 80% germination occurs between the 12th and the 20th day after sowing.Different from previously reported, the low vigor and the nuts germination delay observed in this study, may also be attributed to genetic uneven of the nuts, as these come from a heterogeneous population and uneven, which may have contributed to the results.
The results of this study showed that fungal associated to cashew nuts significantly damage the normal development and quality of seedlings, as well as serve as the initial inoculum for future disease outbreaks.

Conclusion
The presence of Aspergillus sp., Cladosporium sp., Penicillium sp. and C. gloeos porioides were observed in most of the analyzed nuts.The pat hogen C. gloeosporioides can be transmitted efficiently to cashew seedlings via nuts.Cashew nuts with a high degree of anthracnose severity lead the emergence of a larger number of symptomatic seedlings.

Figure 2 .
Figure 2. Anthracnose average incidence (A) and disease index (B) of cashew leaves and nuts ( Anacardium occidentale L.).Percentage of anthracnose incidence data transformed in arcsin x/100.Means by the same letter are not significantly different (Tukey, P≥0.05).

Table 1 .
Relationship betw een the infection severity in cashew nuts ( Anacardium occidentale L.) and trans mission of cashew anthracnose by Colletotrichum gloeosporioides.