Eur J Plant Pathol (2015) 141:761–768 DOI 10.1007/s10658-014-0576-5
Interaction of Rotylenchulus reniformis and Meloidogyne javanica with mealybug wilt of pineapple, in microplots Thiago de Freitas Ferreira & Ricardo Moreira Souza & Karla Daiana dos Santos Ferreira & Welington Sérgio Silva Idalino
Accepted: 11 December 2014 / Published online: 19 December 2014 # Koninklijke Nederlandse Planteziektenkundige Vereniging 2014
Abstract Mealybug wilt of pineapple (MWP) is a disease complex whose etiology seems to involve the mealybug Dysmicoccus brevipes and pineapple mealybug wilt-associated virus-1, −2 and/or −3. MWP, the reniform and the root-knot nematodes (RN, RKN) are major problems of pineapple crop worldwide. Although these nematodes are often found in MWP-affected plantations, so far, no study has investigated possible interactions among these pests causing symptoms and affecting the development of pineapple plants. Pineapple plantlets free of diseases and pests (Vitória cultivar) were inoculated with i) RN; ii) RKN; iii) viruliferous mealybugs; iv) RN and viruliferous mealybugs; and v) RKN and viruliferous mealybugs. Uninoculated plants served as controls. After inoculation, the plants were transplanted into microplots, where shoot symptoms and growth were assessed at 9 and 16 months after inoculation. RN, RKN and MWP induced distinct shoot symptoms, which appeared much worse in coinoculated plants, including the collapse and death of nearly half of the experimental plants at 16 months. RKN, RN and MWP, alone or in combination, reduced the fresh weight of the root system up to 88 % and shoot weight up to 68 %. The interaction among these pests was additive. The results of this experiment emphasize
T. d. F. Ferreira : R. M. Souza (*) : K. D. d. S. Ferreira : W. S. S. Idalino Grupo de Pesquisa em Nematologia, Universidade Estadual do Norte Fluminense Darcy Ribeiro/CCTA/LEF, Campos dos Goytacazes, RJ, Brazil e-mail:
[email protected]
the need for further studies conducted in commercial plantations to understand the interactions among RKN, RN and MWP, and their relative importance, since these are often misdiagnosed and consequently poorly managed. Keywords Reniform nematode . Root-knot nematode . Dysmicoccus brevipes . Pineapple mealybug wiltassociated virus . Ananas comosus
Introduction In Brazil, pineapples (Ananas comosus L. Merril) are cultivated in several states, especially by smallholders. In recent years the pineapple agribusiness has expanded considerably, becoming the economic mainstay of many regions and families (IBGE, 2010). The pineapple plant is affected by a wide variety of pests and diseases, with plant parasitic nematodes (PPNs) and mealybug wilt of pineapple (MWP) being the main problems (Lacerda et al. 2009; Rohrbach and Apto 1986; Sipes et al. 2005). Surveys in the main production areas of Brazil have indicated that Meloidogyne javanica (Treub, 1885) Chitwood, 1949 and Rotylenchulus reniformis Linford and Oliveira 1940 are among the most frequent and damaging PPNs (Cavalcante et al. 1984; Costa et al. 1998; Manso et al. 1994; Zem and Reinhardt 1978). Yield losses due to R. reniformis vary from 60 to 74 % in the first harvest and around 40 % in the second (“ratton”) harvest, which is obtained from plantlets that develop between the
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leaves of the declining “mother” plant. M. javanica can cause a reduction of 10 % in the development of plants (Costa and Matos 2000; Sipes et al. 2005). In Hawaii (USA), MWP is caused by the association of pineapple mealybug wilt-associated virus (PMWaV)-2 and its vector, the mealybug Dysmicoccus brevipes (Cockerell, 1983) (Sether et al. 2010). In Australia, PMWaV-1, −2 and −3, but not PMWaV-5 or the mealybug, seem to be involved in MWP, while in Cuba pineapple plants affected by MWP have been found to carry PMWaV-1, −2 and −3, individually or in combination (Gambley et al. 2008; Hernandez-Rodriguez et al. 2014). In Brazil, studies in germplasm collections have revealed PMWaV-1, −2 and −3 in symptomatic pineapple plants (Peron 2011; Santos et al. 2011). Yield losses due to MWP may exceed 50 % depending on the age of the plant at the beginning of the disease (Sether and Hu 2002). Some reports indicate that plants parasitized by D. brevipes, R. reniformis or M. javanica, or affected by MWP, can present yellowish or reddish leaves, wilted leaf tips, symptoms of nutritional deficiency, lack of response to fertilizers and stunted fruits (Lacerda et al. 2009; Sanches 2005; Sipes et al. 2005). Therefore, the similarity of symptoms can make diagnosis difficult and reduce the efficacy of control or management practices. Nematode interactions with other plant pathogens have been summarized elsewhere (Abawi and Chen 1998; Back et al. 2002). In pineapple, Sipes et al. (2002) observed an additive interaction between R. reniformis and PMWaV-1, in the absence of mealybugs, in reducing plant growth. Costa and Matos (2000) suggested that Pratylenchus brachyurus Filipjev and Stekhoven, 1941 and D. brevipes, without confirming that the insects carry virus, act synergistically to reduce plant growth and production, but their experimental design and results do not prove this claim. So far, the only interactions between PPNs and MWP have been found for P. brachyurus or Helicotylenchus sp. causing additive damage (Ferreira et al. 2014). In Brazil, R. reniformis, M. javanica and MWP often occur concomitantly in pineapple plantations, and these pests are not easily distinguished from each other based on the symptoms they cause. This prevents proper diagnosis and, consequently, effective management. Hence, this work aimed to characterize the symptoms induced by these pests, individually and combined, as well as to assess their damage to plants.
Eur J Plant Pathol (2015) 141:761–768
Material and methods Plantlets of pineapple cultivar Vitória, c. 15 cm tall and propagated in vitro were transplanted individually into plastic pots containing 20 l of washed riverbed sand. The plantlets grew in a greenhouse for 16 weeks, and were fertilized every 2 weeks accordingly to recommendations for the crop (Ramos et al. 2009). When the plantlets reached 30 cm in height, the pots were removed from the greenhouse and partially buried in the soil to establish microplots. At microplot establishment, 25 g/pot of slow-release fertilizer was applied (Osmocote®, 15-10-10). This fertilization was repeated every 5 months until the end of the study, 16 months later on. In periods of low rainfall (July and December 2012), the plants were irrigated immediately after fertilization. The only treatments applied were manual removal of weeds within pots and manual grass cutting in the whole experimental area. The frequency and intensity of rainfall and air temperature were monitored with Watchdog® sensors and datalogger. Thirty days after the microplots were established, the plants were subdivided into treatments (T): T1: control plants free of nematodes and D. brevipes; T2: plants inoculated with D. brevipes; T3: plants inoculated with R. reniformis; T4: plants inoculated with R. reniformis and D. brevipes; T5: plants inoculated with M. javanica; T6: plants inoculated with M. javanica and D. brevipes. There were eight replicates (one plant /pot) per treatment, with a total of 48 plants. The experimental design was randomized blocks within two main microplots of 24 plants each and an area of c. 30 m2 (8.5×3.5 m). The microplot with the 24 plants inoculated with D. brevipes was located about 500 m away from the other microplot to avoid cross contamination. Also, all pots in this microplot were surrounded by formicide Mirex-s® to avoid mealybug’s spread by ants. In each microplot, the plants were arranged in three rows as practiced by Brazilian farmers: two plant rows 40 cm apart spaced 1.2 m from the third row. Within each row there were eight plants, about 40 cm apart. The eight blocks were arranged transversally to the rows. In each block containing three pots, the treatments were randomly assigned. D. brevipes was obtained from pineapple plantations with a high incidence of MWP in the nearby municipality of São Francisco do Itabapoana, and maintained on pineapple plants in a greenhouse. To confirm the incidence of PMWaV in the mealybugs and, later in the
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experimental plants, a RT-PCR test according to Sether et al. (2005) was conducted. For the inoculation of experimental pineapple plants, fragments of the leaves containing D. brevipes were cut to give 25 D. brevipes/ plant, and these pieces were distributed on the axils of the plants. R. reniformis and M. javanica were obtained from commercial pineapple plantations in São Francisco do Itabapoana. R. reniformis was identified based on the key provided by Siddiqi (2000). The nematodes were multiplied on pineapple plants in a greenhouse before inoculation. M. javanica was identified by isoenzyme electrophoresis of females (Esbenshade and Triantaphyllou 1985). For the inoculation of experimental plants, R. reniformis was extracted from pineapple roots in a Baermann funnel and transferred to glass tubes containing 10 mL of water. With a micropipette, 50 specimens were inoculated in the root system of each plant. Second-stage juveniles (J2) of M. javanica were obtained by processing 200 cm3 of soil (Jenkins 1964) and an aliquot of 10 ml of water containing 500 J2 was inoculated on each plant. Eight months after inoculation (M.A.I.), three perforations were made around each plant with a probe to collect 150 cm3 of soil and root fragments. The soil was mixed and the roots separated and weighed, and recorded as fresh root weight/sample. The nematodes were extracted from the soil (Jenkins 1964), counted and recorded as population density per 150 cm3 of soil. The roots were placed in a Baermann funnel for 48 h to obtain the nematodes. After counting, they were recorded as population density per 10 g of root. Dleaves - the youngest physiologically mature ones, the fourth from the plant apex - were collected in the morning, weighed and measured for length and width. The plants were induced to flower at 9 M.A.I., when the D-leaf reached on average 57 cm in length, by applying 50 ml/plant of aqueous solution of 0.1 % Etrel, 2 % urea and 0.035 % calcium hydroxide (Veloso et al. 2001). At 16 M.A.I., the nematode population density per 150 cm3 of soil and per 10 g of root (using the same sampling methods as before), and fresh weight, length and width of D-leaf were determined. The plants were then cut to soil level for measurement of the fresh weight of the shoot. After that, the pots were lifted and the soil was carefully washed off the root system under tap water. To avoid losing rootlets, the water was collected in a bucket and passed through a sieve. The fresh weight of the root system was then assessed.
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The whole experiment was repeated once, with a time lag of about 15 days between experimental settings. The second experiment had the same design as the first with the microplots with and without D. brevipes located adjacent to the previous ones. For statistical analysis, the data collected were tested for homogeneity of variances (Cochran and Bartlett tests) and for normality of errors (Lilliefors test), at 5 % probability, and no transformation was required (Fundação Arthur Fundação Arthur 2007). Afterwards, the data were submitted to ANOVA with time being one of the factors and the F-test revealed no significance (P >0.05). Thus, data from both experiments were then combined, and treatment means were compared by the Tukey test (P 0.05) independently of whether plants were affected by MWP. At 8 M.A.I., MWP-affected plants had reduced turgidity of the leaves, and plants parasitized by R. reniformis had chlorotic leaves. In addition to these symptoms, MWP-affected plants parasitized by R. reniformis also revealed chlorotic spots (Fig. 2a). The leaves of MWP-affected plants parasitized by M. javanica also had reduced turgidity. There was a decrease (P
