Organic fertilization influences nematode diversity and maturity index in coffee tree plantations using an agroforestry system.

In conventional coffee farming, soil fauna can be negatively affected by the intensive management practices adopted and the use of an agroforestry system (AFS) is an alternative to reduce these impacts. In coffee AFS, soil nutrition is provided mainly using organic fertilizers. This soil management favors the microbiota and can alter the population dynamics of some organisms. Our objective was to evaluate the effect of organic fertilizers on the nematode community in coffee AFS and to determine their impact on soil ecology. Soil samples were collected from three coffee AFS and a nearby Atlantic rainforest fragment. Nematodes were extracted from the samples and identified to the genus. The identified populations were compared using several community and diversity indices to determine the environmental conditions of the systems under evaluation. No differences in total abundance among nematode communities were found in the four areas evaluated. Regarding trophic groups, the coffee AFS treated with either cow manure or poultry litter favored the trophic group of bacterivores. Plant-parasitic nematodes were more abundant in soils of both the naturally fertilized coffee AFS and the Atlantic rainforest fragment. The maturity and structural indexes indicated that the Atlantic rainforest fragment and the naturally fertilized coffee AFS had similar ecological functions. On the other hand, soils fertilized with cow manure were less diverse, had higher dominance in the community, and showed less ecological stability. The nematode communities found in the AFS were similar to those seen in the forest fragment indicating that is possible to produce coffee sustainably without negatively affecting soil quality.

Intensive sn class="Chemical">oil maclass="Chemical">nagemeclass="Chemical">nt practices, such as deep turclass="Chemical">nover, use of syclass="Chemical">nthetic fertilizers aclass="Chemical">nd pesticides may lead to aclass="Chemical">n imbalaclass="Chemical">nce iclass="Chemical">n sclass="Chemical">n class="Chemical">oil biodiversity and negatively affect the functionality of agroecosystems (Wall et al., 2015). Hence, some farmers have begun managing their crops in agroforestry systems (AFS) using agroecological and organic approaches to reduce the environmental impact caused by intensive agriculture. Incorporation of different tree species into coffee plantations may promote social, economic, and environmental benefits as this contributes to production diversity, increased family income, and increased organic matter in the soil (Jackson et al., 2012).

In coffee AFS, sn class="Chemical">oil class="Chemical">nutritioclass="Chemical">n is provided from orgaclass="Chemical">nic fertilizers such as class="Chemical">n class="Species">cow manure, poultry litter and plant residues when necessary. The proper incorporation of these fertilizers improves soil quality and microbial activity (Jannoura et al., 2014). These fertilizers can also change the population dynamics of some organisms such as nematodes (Jannoura et al., 2014; Falkowski et al., 2019).

Sn class="Chemical">oil class="Chemical">nematodes caclass="Chemical">n have differeclass="Chemical">nt habits aclass="Chemical">nd are classified as either free-liviclass="Chemical">ng (bacterivores, fuclass="Chemical">ngivores, predators, aclass="Chemical">nd omclass="Chemical">nivores) or placlass="Chemical">nt-parasites (Ferris, 2010). These orgaclass="Chemical">nisms play aclass="Chemical">n importaclass="Chemical">nt ecological role iclass="Chemical">n regulaticlass="Chemical">ng the sclass="Chemical">n class="Chemical">oil microbiota, mineralization, and nutrient cycling (Bongers and Ferris, 1999). Nematodes are sensitive to changes in mulching and respond rapidly to agricultural practices such as fertilization (Ferris, 2010; Oka, 2010). Studies on the diversity of nematodes in agricultural areas have resulted in a growing interest in this field as these organisms can act as bioindicators for agroecosystems (Neher, 2001).

Depending on the fertilizer used, the availability of sn class="Chemical">oil class="Chemical">nutrieclass="Chemical">nts is altered aclass="Chemical">nd this reflects oclass="Chemical">n the class="Chemical">nematode commuclass="Chemical">nity (Yeates et al., 2009; Zhaclass="Chemical">ng et al., 2019). Orgaclass="Chemical">nic fertilizatioclass="Chemical">n caclass="Chemical">n provide class="Chemical">nutrieclass="Chemical">nts for the developmeclass="Chemical">nt of bacterial-feediclass="Chemical">ng class="Chemical">nematodes aclass="Chemical">nd reduce the class="Chemical">numbers of some class="Chemical">n class="Disease">plant-parasitic nematode species (Renco and Kovacik, 2012). Although the effects of fertilization on the abundance and diversity of soil nematodes have been widely studied, the impact of organic fertilization on complex agroecosystems, such as AFS, is unknown (Zhang et al., 2019). Thus, the objectives of this study were to (1) evaluate the effect of soil fertilization using either poultry litter, cow manure, or plant residues on nematode communities in coffee AFS and (2) determine the level of anthropic disturbance in the agroecosystem by comparing it to that of a nearby undisturbed rainforest fragment.

Materials and methods

Study site

This work was carried out at a rural property within the municipality of Araponga, Zona da Mata in Minas Gerais state, Brazil (20º 38´ 39.76´´ S, 42º 3´ 0.27´´W), located at an altitude of 1,315 m. The region’s climate is mesothermal and annual rainfall varies from 1,280 mm to 1,800 mm. The terrain is mountainous; declivity is between 20 and 45%, with prevalence of n class="Chemical">latosols. This rural property had three coffee placlass="Chemical">ntatioclass="Chemical">ns (class="Chemical">n class="Species">Coffea arabica L. cv. Red Catuaí) using the agroforestry system and was close to an Atlantic rainforest fragment.

History and characterization of the study areas

In the 1980s, this rural property was used for corn and bean cultivation and n class="Species">cattle raisiclass="Chemical">ng. At that time, burclass="Chemical">niclass="Chemical">ng practices were frequeclass="Chemical">nt. The coffee placlass="Chemical">ntatioclass="Chemical">ns begaclass="Chemical">n iclass="Chemical">n 1999. Chemical fertilizers were applied to the coffee tree placlass="Chemical">ntatioclass="Chemical">ns betweeclass="Chemical">n 1999 aclass="Chemical">nd 2001. After that, chemical fertilizers were gradually replaced by orgaclass="Chemical">nic fertilizers. Iclass="Chemical">n 2003, other species of trees were iclass="Chemical">ncorporated iclass="Chemical">nside the coffee placlass="Chemical">ntatioclass="Chemical">ns, aclass="Chemical">nd siclass="Chemical">nce theclass="Chemical">n, the traclass="Chemical">nsitioclass="Chemical">n from solely coffee placlass="Chemical">ntatioclass="Chemical">ns to AFS begaclass="Chemical">n. Farmers placlass="Chemical">nted trees, especially fruit trees, raclass="Chemical">ndomly growclass="Chemical">n withiclass="Chemical">n the coffee placlass="Chemical">ntatioclass="Chemical">ns. Other maclass="Chemical">nagemeclass="Chemical">nt chaclass="Chemical">nges also occurred: farmers stopped weediclass="Chemical">ng spoclass="Chemical">ntaclass="Chemical">neous growiclass="Chemical">ng placlass="Chemical">nts, started tilliclass="Chemical">ng the sclass="Chemical">n class="Chemical">oil, and stopped using pesticides. Currently, two coffee plantations are certified as organic by BCS Öko-Garantie, a German certification organization. One of the plantations is fertilized using poultry litter and the other one with cow manure. In a third plantation, a natural farming system (only plant residues are added to the tilled soil) was adopted and the crop has been certified by the Shumei Japanese organization of natural farmers.

The experimental design was carried out in coffee plantations using three different organic fertilizer treatments (natural fertilizer (NF), poultry litter (PL), n class="Species">cow maclass="Chemical">nure (CM)) aclass="Chemical">nd iclass="Chemical">n a fragmeclass="Chemical">nt forest (FF). The 25,000 square meters of Atlaclass="Chemical">ntic forest fragmeclass="Chemical">nt is coclass="Chemical">nsidered as a remclass="Chemical">naclass="Chemical">nt of the forest aclass="Chemical">nd does class="Chemical">not preseclass="Chemical">nt receclass="Chemical">nt sigclass="Chemical">ns of aclass="Chemical">nthropogeclass="Chemical">nic disturbaclass="Chemical">nces, except for activities iclass="Chemical">nvolviclass="Chemical">ng removal of falleclass="Chemical">n braclass="Chemical">nches that were used for fertiliziclass="Chemical">ng class="Chemical">natural coffee placlass="Chemical">ntatioclass="Chemical">ns.

The naturally fertilized coffee plantation was comprised of an area of 8,000 square meters, with 2.3 × 1.2 meters spacing between coffee trees. In this system, coffee trees were last pruned in 2011. Such plantations are mostly cultivated together with the following plant species: n class="Species">banana (class="Chemical">n class="Species">Musa sp.); capoeira-branca (Solanum argenteum); royal palm (Archontophoenix cunninghamiana) and pinto peanut (Arachis pintoi). Plant residues already present in coffee plantations are used as fertilizers, including banana pseudostem, capoeira-branca trunks, pinto peanuts, and leaf litter from the nearby forest fragment. Fertilization is performed by placing the plant material in a circle on the soil around the coffee tree, and the coverage is based on the size of the crown. This is performed once in November and again 45 days later. In total, approximately 70 tonnes ha-1 of residues are used per year in the plantations.

The coffee plantation fertilized with poultry litter comprised an area of 4,000 square meters, with 1.5 × 3.0 m plant spacing. Fertilization was performed by placing the fertilizer on the sn class="Chemical">oil iclass="Chemical">n a circle arouclass="Chemical">nd the coffee tree, aclass="Chemical">nd coverage was based oclass="Chemical">n the size of the crowclass="Chemical">n. This was carried out oclass="Chemical">nce iclass="Chemical">n December aclass="Chemical">nd agaiclass="Chemical">n after 45 days after the first applicatioclass="Chemical">n. A total of 44.5 toclass="Chemical">nclass="Chemical">nes ha-1 of composted poultry litter, obtaiclass="Chemical">ned from class="Chemical">neighboriclass="Chemical">ng farms, was used per year iclass="Chemical">n this placlass="Chemical">ntatioclass="Chemical">n. To compost poultry litter, this material was assembled iclass="Chemical">n layers iclass="Chemical">nterspersed with maclass="Chemical">nure aclass="Chemical">nd coffee beaclass="Chemical">n peels from the iclass="Chemical">nteclass="Chemical">nded placlass="Chemical">ntatioclass="Chemical">n. The material was moisteclass="Chemical">ned at least oclass="Chemical">nce a week aclass="Chemical">nd turclass="Chemical">ned iclass="Chemical">n every 30 days. The eclass="Chemical">ntire process takes 90 days. The predomiclass="Chemical">naclass="Chemical">nt tree species iclass="Chemical">n the area are “guatambu” (Aspidosperma polyclass="Chemical">neurum); “capoeira-braclass="Chemical">nca” (class="Chemical">n class="Species">Solanum argenteum) and banana (Musa sp.). Except for the banana trees that are planted around the coffee tree plantations as a physical wind barrier, the other species were randomly distributed in the plantations.

The coffee tree plantation fertilized using composted n class="Species">cow maclass="Chemical">nure comprised aclass="Chemical">n area of 15,000 square meters, with 3.0 × 1.5 m placlass="Chemical">nt spaciclass="Chemical">ng. Fertilizatioclass="Chemical">n was performed by placiclass="Chemical">ng the fertilizer oclass="Chemical">n the sclass="Chemical">n class="Chemical">oil in a circle around the coffee tree, and coverage was based on the size of the crown. This was performed once in December and again after 45 days after the first application. In total, approximately 13.2 tonnes ha-1 of composted cow manure from the rural property was used per year in the plantation. For composting, the manure was heaped in a pile, moistened every week, and turned every 30 days. The entire process takes 90 days. In this area, the main tree species were avocado (Persea gratissima) and banana (Musa sp.).

Soil analyses

Physical analyses of sn class="Chemical">oil total porosity, microporosity, macroporosity, sclass="Chemical">n class="Chemical">oil moisture, and chemical analyses of soil macronutrients, organic matter, C:N ratio, and pH from the four areas evaluated were carried out. In each experimental plot were samples were collected from the soil layer at a depth of 0–10 cm with the aid of a volumetric ring. The methods used in the physical and chemical soil analyses were according to Donagema et al. (2011). The collection of soil samples for physical and chemical analyses was simultaneous with the collection of samples for nematological analyses.

Sampling, extraction, and identification of nematodes

Sn class="Chemical">oil samples for estimatioclass="Chemical">n of class="Chemical">nematode populatioclass="Chemical">ns were collected from the three coffee placlass="Chemical">ntatioclass="Chemical">ns aclass="Chemical">nd the Atlaclass="Chemical">ntic forest fragmeclass="Chemical">nt. For each fertilizatioclass="Chemical">n treatmeclass="Chemical">nt, five coffee trees were raclass="Chemical">ndomly selected aclass="Chemical">nd the sclass="Chemical">n class="Chemical">oil at the base of the plant was sampled in a zigzag pattern. In each area, five soil samples were collected at a depth of 0–20 cm in September 2015 using a volumetric ring with a 2.5-cm diameter. A total of 12 samples were collected and homogenized as a mixed sample for each coffee tree selected (Huising et al., 2008) (Figure 1). Samples were then transferred into plastic bags, sealed, and labeled for subsequent extraction and identification of nematodes.

Figure 1:

Sampling method for nematodes present in the soil in each coffee plantation.

Sampling method for nematodes present in the sn class="Chemical">oil iclass="Chemical">n each coffee placlass="Chemical">ntatioclass="Chemical">n.

Extraction of nematodes was performed using the flotation method with a n class="Chemical">sucrose solutioclass="Chemical">n (Jeclass="Chemical">nkiclass="Chemical">ns, 1964). Sclass="Chemical">n class="Chemical">oil samples (100 cc) were first passed through a 2-mm mesh sieve to remove stones and roots. Sieved soils were then placed in a beaker containing 1,000 mL of water, and suspensions were manually stirred for a minute. Then, stirring was interrupted for 20 s so that thicker particles could settle and the supernatants were then transferred to a 400-mesh sieve. The retained material was then washed and transferred to Falcon tubes (50 mL) and centrifuged for 5 min at 1,008 × g, and the supernatants were discarded. Then, a sucrose solution (460 g of sucrose per liter of water) was added to the tubes, and the mixture was centrifuged for one minute at 112 × g. Suspensions were then passed through a 400-mesh sieve and washed under running water to remove the sucrose. Nematodes retained by the sieve were then transferred to Falcon tubes (50 mL) containing 10 mL of water. The tubes were kept in an incubator for 5 min at a temperature of 64°C, resulting in the death of the nematodes; however, the nematodes were not deformed by this process. Then, they were fixed by the addition of 1 mL of formaldehyde solution and stored at 4°C in a refrigerator.

Suspensions containing nematodes were pipetted into flat glass Petri dishes (60 diameter × 15-mm height) that were then placed under a binocular stereoscope (Bel Photonics®, model SZ) to count the nematodes. Nematodes were picked up using a needle and transferred to microscope slides (Precision Glass®, 26 × 76 mm) containing a drop of n class="Chemical">water + class="Chemical">n class="Chemical">formaldehyde at a ratio of 10:1.

A double layer of matt varnish (Acrilex®) was applied to each slide to fix the coverslips in place (Paiva et al., 2006). To seal the space between the slide and coverslip, a double layer of colorless nail polish (Risqué®) was then applied. Nematodes were observed using an optical microscope (40 × objective) (Motic®-A210 model) and identified to the genus level using standard keys (Andrássy, 2005; Chaves et al., 1995; Jairajpuri and Wasim, 1992; Siddiqi, 2000), and classified into five trophic groups based on feeding habits: bacterivores, plant-parasites, fungivores, omnivores, and predators.

Ecological parameters and indices of communities and ecosystems

The effect of fertilization on the nematode communities was assessed by calculating total abundance, trophic abundance, and relative abundance. Trophic abundance is the abundance of nematodes within the different trophic groups. Relative abundance was calculated as the percentage value of the number of genera that belong to a particular taxon or trophic group in relation to the total number of nematodes present in a sample using the following equation: p  = (n/N) × 100, wherein n was the number of nematodes from each genus and N was the total number of nematodes in each sample (Cares and Huang, 2008).

To compare the diversity and dominance between the areas, the Shannon (H´) and Simpson (Ds) indexes were calculated. The Shannon index applies equal weight to rare and abundant genus (Shannon, 1948) using the equation H´ = −∑ p × LN (p), wherein p is the relative abundance and LN is the logarithmic function. The Simpson index measures the probability of two randomly selected nematodes from a sample belonging to the same species. This index is calculated using the equation Ds = ∑ (p)², wherein p was the relative abundance of nematodes in a sample (Simpson, 1949).

The specific indexes for nematodes that were developed by Bongers (1990), Maturity (MI), Maturity 2–5 (MI 2–5), and Plant Parasites (PPI) indexes, and by Ferris (2010), Basal (BI), enrichment (EI), and structure (SI) indexes were calculated to obtain the environmental conditions of the areas studied.

To calculate the maturity indexes, all sn class="Chemical">oil class="Chemical">nematodes except placlass="Chemical">nt-parasitic oclass="Chemical">nes were coclass="Chemical">nsidered. MI 2–5 was based oclass="Chemical">n the calculatioclass="Chemical">ns for the class="Chemical">nematode groups that had a c-p value betweeclass="Chemical">n 2 aclass="Chemical">nd 5; therefore, placlass="Chemical">nt-parasitic aclass="Chemical">nd class="Chemical">nematodes with a c-p value of 1 are excluded. The Maturity Iclass="Chemical">ndex is coclass="Chemical">nsidered a measure of eclass="Chemical">nviroclass="Chemical">nmeclass="Chemical">ntal disturbaclass="Chemical">nce, aclass="Chemical">nd low MI values iclass="Chemical">ndicate disturbed aclass="Chemical">nd eclass="Chemical">nriched eclass="Chemical">nviroclass="Chemical">nmeclass="Chemical">nts while high MI values iclass="Chemical">ndicate stable eclass="Chemical">nviroclass="Chemical">nmeclass="Chemical">nts (Boclass="Chemical">ngers, 1990). Due to the fuclass="Chemical">nctioclass="Chemical">nality of class="Chemical">n class="Disease">plant-parasitic nematodes in the agroecosystem, this group is used to calculate the level of anthropic disturbance using PPI (Bongers, 1990). The equation used to calculate MI, MI 2–5 and PPI is ∑[v(i) × pi], where v(i) corresponds to the c-p value of the nematode family and pi the relative abundance of that family in the sample (Bongers, 1990).

The Basal Index (BI), Enrichment Index (EI), and Structure Index (SI) are based on the importance of functional guilds of nematodes as indicators and are descriptors of food web conditions. Basal food webs are those which are diminished due to stress, scarce availability of resources, contamination, or other harsh environmental conditions. Nematodes present in these food webs are represented by bacterial and fungal feeding taxa from the n class="Chemical">c-p2 class of the MI. Eclass="Chemical">nriched food webs are those with high availability of resources due to the occurreclass="Chemical">nce of a disturbaclass="Chemical">nce eveclass="Chemical">nt. Opportuclass="Chemical">nistic bacterial feediclass="Chemical">ng class="Chemical">nematodes from the c-p1 class are predomiclass="Chemical">naclass="Chemical">nt iclass="Chemical">n these food webs. Fuclass="Chemical">ngal feediclass="Chemical">ng class="Chemical">nematodes from the class="Chemical">n class="Chemical">c-p2 class might increase when more complex resources, such as with a higher C:N ratio, becomes available, or when fungal feeding activity is enhanced in detriment to bacterial feeding activity. Structured food webs are those with more resources available. Nematode taxa from the c-p3 class are present in less structured food webs, while structure in the community will be greater when nematode taxa from the c-p4 and c-p5 classes are present. These indexes were obtained through the NINJA platform (Sieriebriennikov et al., 2014) and to characterize the conditions of the studied areas, the data were plotted according to Ferris (2010).

Statistical analysis

The values obtained for the attributes from the physical and chemical sn class="Chemical">oil aclass="Chemical">nalyses were submitted to aclass="Chemical">nalysis of variaclass="Chemical">nce (oclass="Chemical">ne-way ANOVA), aclass="Chemical">nd wheclass="Chemical">n the results were statistically sigclass="Chemical">nificaclass="Chemical">nt, they were compared usiclass="Chemical">ng the Tukey test (p < 0.05). The chemical attributes for class="Chemical">n class="Chemical">phosphorus (P) and potassium (K) did not follow a normal distribution, therefore the averages obtained were submitted to non-parametric Kruskal–Wallis analysis, and compared using Dunn’s method (p < 0.05).

The calculated abundances, diversity, and dominance measures, and nematode indexes were submitted to analysis of variance, and when the results were statistically significant, they were compared using the Tukey test (p < 0.05). Genera-related abundance did not follow a normal distribution; therefore, the non-parametric Dunnett’s method was used in this case. Statistical analysis was performed using the SigmaPlot® 12.0 (Systat Software, Inc.) software.

Results

The physical attributes of the sn class="Chemical">oils did class="Chemical">not differ amoclass="Chemical">ng the areas (FTP (3,19)  = 1.08, P = 0.38; FMicro (3,19)  = 1.55, P = 0.24; FMacro (3,19)  = 2.16, P = 0.13; FMicro (3,19)  = 2.93, P = 0.06; Table 1). Iclass="Chemical">n relatioclass="Chemical">n to the chemical attributes, pH aclass="Chemical">nd class="Chemical">n class="Chemical">nitrogen did not present any statistical differences (FpH (3,19)  = 1.58, P = 0.23; FN (3,19)  = 1.35, P = 0.29; Table 2). The macro-nutrient phosphorus (P) displayed higher values in PL and CN (H(3,19)  = 17.65, p < 0.001), potassium was found at a higher concentration in PL (H(3,19)  = 16.71, p < 0.001), whilst the level of organic material was higher in FF, PL and NF (F(3,19)  = 20.74, p < 0.001). A higher ratio of C:N was observed in PL and CM (F(3,19)  = 20.24, p < 0.001) (Table 2).

Table 1.

Physical characterization of soil samples collected at a depth of 0–10 cm from the following areas in Araponga, Minas Gerais, Brazil: forest fragment (FF) and coffee plantations naturally fertilized (NF), fertilized with poultry litter (PL) or cow manure (CM).

	Physical attributes (%)
	Textural class	TP	Micro	Macro	SM
NF	Clayey	56.02 ± 1.08	34.79 ± 0.99	21.23 ± 1.55	25.80 ± 1.43
PL	Clayey	59.23 ± 1.68	36.83 ± 0.90	22.40 ± 1.13	27.23 ± 0.94
CM	Clayey	58.51 ± 1.01	33.27 ± 1.22	25.26 ± 1.79	30.12 ± 1.18
FF	Clayey	59.34 ± 1.95	32.12 ± 2.75	27.21 ± 2.59	25.67 ± 1.08

Notes: * Macro, macroporosity; Micro, microporosity; SM, Soil moisture; TP, total porosity.

**Values are means ± s.e.m.; none of the results were significantly different using analysis of variance (p < 0.05).

Table 2.

Chemical characterization soil sampled at a depth of 0–10 cm from the following areas in Araponga, Minas Gerais, Brazil: forest fragment (FF) and coffee plantations, naturally fertilized (NF), poultry litter fertilized (PL) and cow manure (CM) fertilized.

		N	P	K	OM	C:N
	pH	(%)	mg/dm3	mg/dm3	ppm
NF	5.8 ± 0.05ns	0.27 ± 0.01ns	15.4 ± 0.42b	319 ± 1.21ab	17.65 ± 0.52a	25.65 ± 0.23b
PL	5.5 ± 0.08ns	0.28 ± 0.01ns	705.6 ± 6.74a	977 ± 0.79a	17.73 ± 0.63a	48.21 ± 9.60a
CM	5.6 ± 0.09ns	0.27 ± 0.01ns	46.7 ± 0.65a	329 ± 5.37ab	14.48 ± 0.35b	31.01 ± 6.20a
FF	5.7 ± 0.05ns	0.29 ± 0.01ns	3.0 ± 0.05b	60 ± 2.43b	19.46 ± 0.28a	39.01 ± 7.80ab

Notes: C:N = Carbon-nitrogen ratio, K = Potassium, N = total organic nitrogen, OM = Organic matter, P = available Phosphorus.

Values are means ± s.e.m.; ns: Non-significant by analysis of variance. pH, N, OM, and C:N ratio, Tukey Test; P and K Dunn’s method (p < 0.05).

Physical characterization of sn class="Chemical">oil samples collected at a depth of 0–10 cm from the followiclass="Chemical">ng areas iclass="Chemical">n Arapoclass="Chemical">nga, Miclass="Chemical">nas Gerais, Brazil: forest fragmeclass="Chemical">nt (FF) aclass="Chemical">nd coffee placlass="Chemical">ntatioclass="Chemical">ns class="Chemical">naturally fertilized (NF), fertilized with poultry litter (PL) or class="Chemical">n class="Species">cow manure (CM).

Notes: * Macro, macroporosity; Micro, microporosity; SM, Sn class="Chemical">oil moisture; TP, total porosity.

Chemical characterization sn class="Chemical">oil sampled at a depth of 0–10 cm from the followiclass="Chemical">ng areas iclass="Chemical">n Arapoclass="Chemical">nga, Miclass="Chemical">nas Gerais, Brazil: forest fragmeclass="Chemical">nt (FF) aclass="Chemical">nd coffee placlass="Chemical">ntatioclass="Chemical">ns, class="Chemical">naturally fertilized (NF), poultry litter fertilized (PL) aclass="Chemical">nd class="Chemical">n class="Species">cow manure (CM) fertilized.

Notes: C:N = n class="Chemical">Carbon-class="Chemical">n class="Chemical">nitrogen ratio, K = Potassium, N = total organic nitrogen, OM = Organic matter, P = available Phosphorus.

A total of 2139 nematodes were collected and classified into the five trophic groups: plant-parasites, bacterivores, fungivores, omnivores, and predators (Table 3). No differences in total nematode abundance were found when comparing the evaluated areas (F3,19  = 2.26, P = 0.125).

Table 3.

Average values (± standard error) for the total abundance of nematodes per 100 g of dry soil from an Atlantic forest fragment (FF), a naturally fertilized (NF) coffee plantation, poultry litter (PL) fertilized coffee plantation, and a coffee plantation fertilized with cow manure (CM).

Feeding habits	FF	NF	PL	CM
Bacterivores	108 ± 2.73b	170 ± 1.14b	380 ± 7.72a	378 ± 5.83a
Plant-parasites	270 ± 5.03a	287 ± 3.37a	183 ± 3.12b	128 ± 1.28b
Fungivores	84 ± 2.83a	51 ± 5.16a	32 ± 1.12b	13 ± 0.97b
Omnivores	11 ± 0.54ns	9 ± 0.37ns	2 ± 0.41ns	2 ± 0.83ns
Predators	10 ± 0.73ns	9 ± 0.37ns	8 ± 0.24ns	4 ± 0.25ns
Total	483 ± 7.20ns	526 ± 5.63ns	605 ± 9.09ns	525 ± 4.59ns

Notes: Values followed by the same letter on the same line did not differ when using the Tukey test (p < 0.05); nsNon-significant by analysis of variance.

Average values (± standard error) for the total abundance of nematodes per 100 g of dry sn class="Chemical">oil from aclass="Chemical">n Atlaclass="Chemical">ntic forest fragmeclass="Chemical">nt (FF), a class="Chemical">naturally fertilized (NF) coffee placlass="Chemical">ntatioclass="Chemical">n, poultry litter (PL) fertilized coffee placlass="Chemical">ntatioclass="Chemical">n, aclass="Chemical">nd a coffee placlass="Chemical">ntatioclass="Chemical">n fertilized with class="Chemical">n class="Species">cow manure (CM).

Regarding the trophic groups, the abundance of n class="Disease">plant-parasitic nematodes was higher iclass="Chemical">n the sclass="Chemical">n class="Chemical">oil from naturally fertilized coffee plantations and the Atlantic forest fragment (F3,19  = 18.58, P = 0.0001) (Table 3). Bacterivorous nematodes were more abundant in plots that had been fertilized with composted poultry litter and cow manure, than plantations were natural fertilizer was used and in soil from the forest fragment (F3,19  = 30.96, P = 0.0001). Fungivorous nematodes were more abundant in soil from the forest fragment and the naturally fertilized plantation than in the other plantations (F3,19  = 9.52, P = 0.001). No differences were found with respect to omnivorous (F3,19  = 4.40, P = 0.019) and predatory nematodes (F3,19  = 1.49, P = 0.254) (Table 3).

We identified 23 nematode genera distributed into 21 families (Table 4). Bacterivorous nematodes from the genus Acrobeles were significantly more abundant in plantations fertilized with n class="Species">cow maclass="Chemical">nure (class="Chemical">n class="Chemical">H3,19  = 9.15, P = 0.02) as well as the genus Rhabditis which was also more abundant in areas where cow manure was used (H3,19  = 17.86, P = <0.001). In the plant-parasitic trophic group, the genus Criconema (H3,19  = 11.64, P = 0.009) and Pratylenchus (H3,19  = 12.86, P = 0.005) were more abundant in the soil from the forest fragment and naturally fertilized coffee plantations. The genus Helicotylenchus was found in the highest abundance in naturally fertilized soil from coffee plantations (H3,19  = 10.57, P = 0.014). The highest relative abundance of the genus Aphelenchus was found in soil from the forest fragment (H3,19  = 13.65, P = 0.003).

Table 4.

Mean relative abundance of nematode genera found in soil samples from an Atlantic forest fragment (FF), a naturally fertilized (NF) coffee plantation, a poultry litter (PL) fertilized coffee plantation, and cow manure (CM) fertilized coffee plantation.

Families	Genera	FF	NF	PL	CM
Bacterivores		Areas
Alaimidae	Alaimus	0.01ns	0.02ns	0.00ns	0.00ns
Bunonematidae	Bunonema	0.00ns	0.00ns	0.01ns	0.00ns
Cephalobidae	Acrobeles	0.09b	0.10b	0.10b	0.18a
	Cephalobus	0.05ns	0.04ns	0.02ns	0.02ns
Diplogastridae	Diplogaster	0.00ns	0.00ns	0.03ns	0.01ns
Panagrolaimidae	Panagrolaimus	0.00ns	0.01ns	0.03ns	0.01ns
Plectidae	Plectus	0.01ns	0.01ns	0.02ns	0.02ns
	Wilsonema	0.00ns	0.01ns	0.00ns	0.01ns
Prismatolaimidae	Prismatolaimus	0.01ns	0.01ns	0.00ns	0.00ns
Rhabditidae	Rhabditis	0.05b	0.12b	0.38a	0.49a
Teratocephalidae	Teratocephalus	0.00ns	0.01ns	0.02ns	0.00ns
Plant-parasites		Areas
Anguininae	Ditylenchus*	0.03ns	0.02ns	0.04ns	0.01ns
Criconematidae	Criconema	0.15a	0.08a	0.02b	0.01b
Hoplolaimidae	Helicotylenchus	0.21b	0.35a	0.20b	0.17b
Longidoridae	Longidorus	0.03ns	0.01ns	0.02ns	0.02ns
Pratylenchidae	Pratylenchus	0.07a	0.04a	0.01b	0.00b
Trichodoridae	Trichodorus	0.02ns	0.02ns	0.00ns	0.00ns
Tylenchidae	Tylenchus	0.05ns	0.03ns	0.04ns	0.02ns
Fungivores		Areas
Aphelenchidae	Aphelenchus	0.14a	0.07b	0.05b	0.02b
Diphtherophoridae	Diphtherophora	0.02ns	0.01ns	0.00ns	0.00ns
Tylencholaimellidae	Tylencholaimellus	0.01ns	0.01ns	0.00ns	0.00ns
Onivores		Areas
Dorylaimidae	Dorylaimus	0.03ns	0.02ns	0.00ns	0.00ns
Predator		Areas
Mononchidae	Mononchus	0.02ns	0.02ns	0.01ns	0.01ns

Notes: Values followed by the same letter on the same line did not differ when using Dunnett’s method (p < 0.05).

*Ditylenchus specimens found in soil can be plant-parasitic and/or fungivorous.

Mean relative abundance of nematode genera found in sn class="Chemical">oil samples from aclass="Chemical">n Atlaclass="Chemical">ntic forest fragmeclass="Chemical">nt (FF), a class="Chemical">naturally fertilized (NF) coffee placlass="Chemical">ntatioclass="Chemical">n, a poultry litter (PL) fertilized coffee placlass="Chemical">ntatioclass="Chemical">n, aclass="Chemical">nd class="Chemical">n class="Species">cow manure (CM) fertilized coffee plantation.

*Ditylenchus specimens found in sn class="Chemical">oil caclass="Chemical">n be placlass="Chemical">nt-parasitic aclass="Chemical">nd/or fuclass="Chemical">ngivorous.

Regarding diversity and dominance of the studied areas, significant differences were detected both in the Shannon (F3,19  = 12.17, P = <0.001) and Simpson (F3,19  = 82.36, P = <0.001) indexes. In the plantation fertilized with the n class="Species">cow maclass="Chemical">nure, the diversity was lower (1.95) aclass="Chemical">nd domiclass="Chemical">naclass="Chemical">nce was greater (0.33) thaclass="Chemical">n other areas studied (Table 5).

Table 5.

Average values (± standard error) of Shannon and Simpson indices of nematode communities in soil samples from an Atlantic forest fragment (FF), a naturally fertilized (NF) coffee plantation, a poultry litter (PL) fertilized coffee plantation, and cow manure (CM) fertilized coffee plantation.

	Areas evaluated
Diversity indexes	FF	NF	PL	CM
Shannon (H’)	2.34 ± 0.03a	2.24 ± 0.03a	2.09 ± 0.04a	1.95 ± 0.06b
Simpson (Ds)	0.14 ± 0.002b	0.17 ± 0.006b	0.20 ± 0.002b	0.33 ± 0.002a

Notes: Values followed by the same letter on the same line did not differ when using the Tukey test (p < 0.05).

Average values (± standard error) of Shannon and Simpson indices of nematode communities in sn class="Chemical">oil samples from aclass="Chemical">n Atlaclass="Chemical">ntic forest fragmeclass="Chemical">nt (FF), a class="Chemical">naturally fertilized (NF) coffee placlass="Chemical">ntatioclass="Chemical">n, a poultry litter (PL) fertilized coffee placlass="Chemical">ntatioclass="Chemical">n, aclass="Chemical">nd class="Chemical">n class="Species">cow manure (CM) fertilized coffee plantation.

The results for the maturity index (MI) showed significant differences (F3,19  = 122.05, P = <0.001, Figure 2A), with higher values in the forest fragment (2.24) and in the naturally fertilized plantation (1.98), whilst lower values were seen in the sn class="Chemical">oils from placlass="Chemical">ntatioclass="Chemical">ns where class="Chemical">n class="Species">cow manure (1.54) and poultry litter (1.45) had been employed. The maturity index 2–5 (MI 2–5) also a showed significant difference (F3,19  = 7.75; P = 0.002, Figure 2B), where the poultry litter fertilized plantation showed a lower value (2.16) than the other areas. Regarding the plant-parasitic nematode index (F3,19  = 1.99; P = 0.157), no differences were observed between the areas (Figure 2C).

Figure 2:

Average values (± standard error) of (a) the maturity, (b) the maturity 2–5, (c) the plant parasitic, (d) the basal, (e) the enrichment, and (f) the structure indexes. Values followed by the same letter did not differ when using the Tukey test (p < 0.05).

Significant differences were detected in the basal index (F3,19  = 79.75, P = <0.001, Figure 2D), enrichment index (F3,19  = 172.65, P = <0.001, Figure 2E), and structure index (F3,19  = 122.05, P = <0.001, Figure 2F). The basal index was highest in the fragment forest, followed by the naturally fertilized plantation and the lowest values were seen in the areas fertilized with n class="Species">cow maclass="Chemical">nure aclass="Chemical">nd poultry litter (Figure 2D). The eclass="Chemical">nrichmeclass="Chemical">nt iclass="Chemical">ndex showed higher values iclass="Chemical">n sclass="Chemical">n class="Chemical">oil from the coffee plantation fertilized with poultry litter, followed by cow manure, naturally fertilized plantation, and the fragment forest (Figure 2E). The forest fragment, natural coffee plantation, and poultry litter plantation had the highest structure index. Coffee plantations fertilized with cow manure had the lowest value, but did not differ from plantations fertilized with poultry litter (Figure 2F).

The faunal profile data, observed in quadrant A, indicated that the plantations fertilized with poultry litter and n class="Species">cow maclass="Chemical">nure preseclass="Chemical">nted a sclass="Chemical">n class="Chemical">oil enrichment profile and this was an indication of an agroecosystems with a high level of ecological disturbance. The naturally fertilized coffee plantation, observed in quadrant B, presents similar food web characteristics to the forest fragment (Figure 3).

Figure 3:

Food web analysis of coffee crops fertilized with cow manure (CM), poultry litter (PL), plant residues (NF), and in a forest fragment (FF). Quadrant A: enriched and structured agroecosystem; quadrant B: mature and structured environment; quadrant C: mature and stable environment, fertile soil; and quadrant D: degraded and depleted soil. (Ferris, 2010).

Food web analysis of coffee crops fertilized with n class="Species">cow maclass="Chemical">nure (CM), poultry litter (PL), placlass="Chemical">nt residues (NF), aclass="Chemical">nd iclass="Chemical">n a forest fragmeclass="Chemical">nt (FF). Quadraclass="Chemical">nt A: eclass="Chemical">nriched aclass="Chemical">nd structured agroecosystem; quadraclass="Chemical">nt B: mature aclass="Chemical">nd structured eclass="Chemical">nviroclass="Chemical">nmeclass="Chemical">nt; quadraclass="Chemical">nt C: mature aclass="Chemical">nd stable eclass="Chemical">nviroclass="Chemical">nmeclass="Chemical">nt, fertile sclass="Chemical">n class="Chemical">oil; and quadrant D: degraded and depleted soil. (Ferris, 2010).

Discussion

The total abundance of nematodes when comparing the coffee AFS and the Atlantic rainforest fragment indicated that there were similarities in the agroecosystem communities. This result was also observed in similar studies (Franco-Navarro and Godinez-Vidal, 2017; McQueen and Treonis, 2020). Even if sn class="Chemical">oil fertility practices are used, these disturbaclass="Chemical">nces are class="Chemical">not eclass="Chemical">nough to chaclass="Chemical">nge the distributioclass="Chemical">n of class="Chemical">nematode commuclass="Chemical">nities iclass="Chemical">n agroecosystems (Mariclass="Chemical">nho et al., 2014). The preseclass="Chemical">nce of the trees iclass="Chemical">n the coffee placlass="Chemical">ntatioclass="Chemical">ns is aclass="Chemical">n importaclass="Chemical">nt factor that coclass="Chemical">ntributes to regulaticlass="Chemical">ng sclass="Chemical">n class="Chemical">oil temperature, reducing exposure of coffee plants to direct sunlight, and, consequently, making the environment more stable and similar to natural ecosystems (Gomes et al., 2016).

Plant-parasitic and bacterivorous nematodes are usually the dominant trophic groups in natural ecosystems and agricultural plantations (Grabau et al., 2019; Hu et al., 2014). They often exhibit a negative relationship in the sn class="Chemical">oil (Liu et al., 2016), because of the release of compouclass="Chemical">nds aclass="Chemical">nd class="Chemical">n class="Chemical">organic acids combined with high nitrogen levels present in fertilizers of animal origin, which inhibit the abundance of plant-parasitic nematodes (Oka, 2010). On the other hand, bacterivores are favored by the incorporation of animal manure in the soil, leading to increased food resources for bacterial feeders, which contribute to the decomposition of organic matter (Jiang et al., 2013).

The abundance of fungivores is favored by organic matter with high concentrations of n class="Chemical">lignin aclass="Chemical">nd class="Chemical">n class="Chemical">cellulose (organic carbon sources) (Liu et al., 2016) (Table 3). These compounds are commonly found in plant residues from the forest litter, including branch and trunk residues. The forest litter used in the soils of naturally fertilized coffee tree plantations contains minor levels of lignin and cellulose because it has been partially decomposed or is mostly comprised of leaf residues. Fungivorous nematodes are sensitive to ammoniacal acids released in the soil by compounds with a low C:N ratio (Oka, 2010), justifying the lower abundance of this trophic group in the soil fertilized by poultry litter and cow manure.

The n class="Disease">genus Rhabditis was preseclass="Chemical">nt iclass="Chemical">n the greatest abuclass="Chemical">ndaclass="Chemical">nce iclass="Chemical">n sclass="Chemical">n class="Chemical">oils fertilized using poultry litter and cow manure, and the higher abundance of Acrobeles was observed in coffee plantation fertilized with cow manure. These genera belong to groups of opportunistic nematodes and rapidly respond to the incorporation of organic fertilizers from animal origin in the soil and are usually more abundant in agricultural areas (Brmež et al., 2018; Carron et al., 2015; Van den Hoogen et al., 2020). The abundance of members of this family contribute to the mineralization and regulation of nutrients such as nitrogen, which is then available for the trees (Ferris et al., 1998; Sãnchez-Moreno et al., 2011).

The genera Criconema is a n class="Disease">plant-parasitic nematode that is very seclass="Chemical">nsitive to eclass="Chemical">nviroclass="Chemical">nmeclass="Chemical">ntal disturbaclass="Chemical">nces, aclass="Chemical">nd its populatioclass="Chemical">n is favored by sclass="Chemical">n class="Chemical">oils covered with plants and in those with a high content of organic matter, such as natural vegetation and agroecosystems with low anthropogenic disturbance (Caixeta et al., 2016). The genus Helicotylenchus, which is abundant in the soil of naturally fertilized coffee plantations and the forest fragment, is usually a very common plant-parasitic nematode and is present at high densities in annual and perennial plantations and natural ecosystems (Tomazini et al., 2008). As expected, considering the source of the residues used to fertilize the soil of the coffee plantations, the results indicated a greater similarity between soils of naturally fertilized plantations and the Atlantic forest fragment. The presence of these plant-parasites, as well as other opportunistic trophic groups, is favored by a high organic matter content and high root density (Ritzinger et al., 2010). The genus Aphelenchus belongs to the fungivores trophic group and is associated with soils containing high recalcitrant organic matter levels, abundant in habitats with advanced stages of ecological succession (Niles and Freckman, 1998; Porazinska et al., 1999).

The crop fertilized with n class="Species">cow maclass="Chemical">nure showed less diversity aclass="Chemical">nd greater geclass="Chemical">nera domiclass="Chemical">naclass="Chemical">nce, for example like Rhabditis. This area had the lowest declass="Chemical">nsity of trees amoclass="Chemical">ng the agroecosystems studied. These results may be associated with the greater availability of litter iclass="Chemical">n the sclass="Chemical">n class="Chemical">oil, which favors the abundance, diversity, and richness of communities of soil organisms (Moço et al., 2009).

MI values close to two, as found in the forest fragment (2.23) sn class="Chemical">oil aclass="Chemical">nd the sclass="Chemical">n class="Chemical">oil from the naturally fertilized coffee (1.98) plantation (Figure 2), indicated that these environments were in an ecological succession stage. Values between one and two, as obtained for the soils of coffee crops fertilized with animal manure (CM  =  1.43, PL = 1.41), indicated that these agroecosystems present high ecological disturbance due to the enrichment of the soil with organic fertilizers (Bongers, 1990; Bongers and Ferris, 1999). The lowest values for MI 2–5 and the basal index were found in crops fertilized with poultry litter, suggesting a food web dominated by bacteria under nutrient enrichment conditions (DuPont et al., 2009). The similarity of the PPI between areas may be associated with the high C/N ratio found in agroecosystems soils. The lower N availability causes plants to produce less root volume, thus reducing the availability of resources for plant-parasitic nematodes (Ugarte et al., 2013).

The SI combined with EI indicated that coffee crops fertilized with poultry litter and n class="Species">cow maclass="Chemical">nure have similar characteristics. Accordiclass="Chemical">ng to Ferris (2010), these agroecosystems have sclass="Chemical">n class="Chemical">oils with moderate disturbance characteristics, enriched with nitrogen, and with a balanced decomposition channel, although with bacterial decomposition. The soil of the forest fragment and the naturally fertilized coffee plantation showed low disturbance, with a food web characteristic of mature soil. The position near quadrant C indicated that the ecosystem is close to an undisturbed environment, with a decomposition channel dominated by fungi (Figure 2). These results also suggest that the soil condition of the crops is associated with the way they are managed. Agroecological management without the use of chemical inputs (fertilizers and pesticides), no soil disturbance, constant employment of organic residues from trees, and management of spontaneous herbaceous vegetation, favored the quality of the soils of agroecosystems (Mulder et al., 2003).

According to the faunal profile results, it can be inferred that the application of animal waste fertilizers stimulated the bacterial channel. Although they are characterized as disturbed agroecosystems, it can be stated that due to the biodiversity present in AFS, these areas in the long term may be closer to quadrant B, which indicates an environment with lower ecological disturbance (Ferris, 2001). Furthermore, the faunal profile confirms the hypothesis that there is a similarity between the naturally fertilized coffee plantation and the Atlantic rainforest fragment.

The nematodes communities present in the sn class="Chemical">oil of the studied areas showed the similarity betweeclass="Chemical">n the agroforestry systems aclass="Chemical">nd the class="Chemical">natural ecosystem (forest). The systems preseclass="Chemical">nted low levels of ecological disturbaclass="Chemical">nce wheclass="Chemical">n compared to forest fragmeclass="Chemical">nts.