Comparative morphological analysis of apple blister mite, Eriophyes mali Nal., a new pest in Serbia

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has been registered recently (R. Petanović, database of Eriophyoidea of Serbia).Among them, only the apple rust mite (A.schlechtendali) is a widely distributed pest.Analyses made by Amrine (personal communication) have shown that A. malivagrans and A. malus are synonyms of A. schlechtendali, while C. aphrastus is synonym of C. baileyi, E. mali Burts is homonym of E. mali Nalepa, and P. mali (Nalepa, 1926) is synonym of E. mali Nalepa.Moreover, E. malimarginemtorquens is probably a valid species, but found very rarely, while Epitrimerus pyri (Nalepa) has been found on apple as an alternate host, and C. malifoliae is a very questionable species.Summarizing the list of species found on apple trees we may conclude that the apple rust mite (A.schlechtendali), apple blister mite (E.mali) and apple erineum mite (P.malinus) are pests of the apple, while E. malimarginemtorquens, C. baileyi, E. pyri, R. schestovici and D. gigantorhynchus can be its pests under certain circumstances.
The apple blister mite (Eriophyes mali Nal.) (Eriophyidae) has been recently registered for the first time in the territory of Serbia.
Distribution of E. mali has so far been recorded in North America, New Zealand, the European part of Russia, South Africa, Zimbabwe, and in many European countries (Belgium, Finland, Czech Republic, Poland, Germany, Hungary, Bulgaria, Slovenia, Bosnia and Herzegovina, and Croatia).
The apple blister mite was described for the first time by Nalepa (1926) as Eriophyes pyri var.mali, a new variety of Eriophyes pyri (Pgst.1857).The mite caused similar symptoms on apple leaves as the pear blister mite on pear leaves.Type locality was not designated, although it has been suspected that it was in Austria (Amrine & Stasny, 1994).Later, Liro and Roivainen (1951) found the mite as the causative agent of blisters on apple leaves in Southern Finland.Blisters were greenish at the beginning and later became brown.The authors changed the mite's taxonomic status and raised it to the rank of species, i.e.E. mali (Nal.)Liro (nov. comb.).Burts (1970) then described a new species on apple, Eriophyes mali Burts, 1970, which can be considered as a junior homonym of E. mali Nal.(Amrine, personal communication).Manson (1984) detected the species in New Zealand.Considering the species to be E. mali Burts, he compared the morphological features of its New Zealand populations with the mites found by Burts in North Western America.Krasinskaya (1960) erroneously assigned the apple blister mite as Aceria and designated as Eriophyes (Aceria) mali.This designation means that the mite belongs to the genus Eriophyes, subgenus Aceria.In her paper, the biology of the mite, and seasonal changes in the density of its females, males, nymphs and eggs were described, as well as the development of blister galls, their transformations from May to August and their relative abundance and distribution on leaves.Natchev (1978) made a list of eriophyoid mites on the apple in Bulgaria.Among other species, he described the appearance of galls of apple blister gall mites and gave a short description of the mite and a semischematic drawing.In his drawing of the prodorsal shield, the scapular setae (sc) are situated at the rear shield margin directed divergently to the rear, which is one of the diagnostic characters of the genus Aceria.The mite was designated as Aceria mali (Nal.).However, the lateral aspect of the anterior part of the mite clearly pointed at the position of sc tubercles in the position characteristic of the genus Eriophyes (Manson, 1984;Ueckermann, 1993).In a taxonomic study of the genus Eriophyes in Africa, Ueckermann (1993) cited the presence of E. mali Burts, giving only a description of symptoms and distinctive characters of the mite as compared to E. pyri.
The species E. mali was considered for a long time as a variety of the pear blister gall mite (E.pyri) because of its morphological similarities and the appearance of identical symptoms, blister galls on both apple and pear leaves.
Blister galls are made by the initial feeding of overwintering adult females.First symptoms of blister galls are small, red or pink swellings on the undersurface of leaves.Continued feeding of mites kills cells, causing the galls to change colour to dark brown or black on pear leaves and light brown on apple leaves.At the end of the vegetation season infested plant tissue becomes completely necrotic.Blister galls which are not infested by the first generation of mites are light green (Burts, 1970).
A detailed analysis of morphological characters of E. mali and E. pyri showed that they differed in the length of lateral, first ventral and second ventral setae.The setae are significantly longer on E. pyri.Additional features that are different in the two species are the body length and the number of opisthosomal annuli.E. mali is shorter and with fewer annuli (Burts, 1970;Manson, 1984).Moreover, Burts (1970) concluded from the experimental results that the species were host specific.Molecular methods (comparison of ITS1 and ITS2 regions of ribosomal DNA sequences) confirmed that the species were different (Kedzior et al., 2006).
As the species was detected for the first time in Serbia, and E. mali is an important pest, the paper presents the morphological characteristics important for species determination, symptoms and intensity of injury.Morphological characteristics of a population of E. mali from Serbia are compared with E. pyri, and with different populations of E. mali from remote regions of the world using literature data: the original description (Nalepa, 1926), and descriptions of a population from the USA (Burts, 1970) and a population from New Zealand (Manson, 1984).

maTErIaLS aND mETHODS
The presence of E. mali was detected in Serbia in an apple orchard in the suburbs of Belgrade (Ledine N 44 0 48'17.2''E 20 0 21' 34.6'') in July of 2013.Mites were extracted using the method of de Lillo ( 2001).Permanent slides were made in Keifer's F medium using a modified method of Keifer (1975).Mites were observed under a phase contrast light optical microscope (Leica DMLS).Morphological characters of mites were measured using the software package IM 1000 (Leica, Germany), following instructions by Amrine and Manson (1996), and de Lillo et al. ( 2010).Thirty-four characters of 10 females were measured.All measurements are given in micrometers (μm) and, unless stated otherwise, refer to the length of structure.Direct measurements and the means were rounded up to the nearest integer.In order to obtain precise qualitative morphological characters, images were taken, using a scanning electron microscope (SEM, JEOL-JSM 6390) at the University of Belgrade, Faculty of Agriculture.Live mites were collected individually with a fine entomological needle from fresh plant parts under stereomicroscope and placed on a SEM holder.
Male: not seen.E. mali closely resembles E. pyri but, according to comparisons made by Burts (1970) and Manson (1984), there are some characters important for their distinction.As it was stressed in Introduction, the most distinctive characters are: length of lateral (c2), first ventral (d) and second ventral setae (e), length of body and the number of opisthosomal annuli.These morphometric characters are longer, and the number of annuli is higher in E. pyri.
The comparison made in our work (Table 1) shows that the most important distinctive character is the number of opisthosomal annuli, which is higher in E. pyri.Comparing E. mali from different localities (Table 2), differences were evidenced between different populations regarding some morphological structures.The E. mali from Serbia is most similar to the E. mali described by Nalepa (1926).Both populations are characterized by almost the same body length, length of first and second legs, second ventral setae (e), and the number of striae on epigynium.E. mali from the USA and New Zealand populations are characterized by shorter body, first and second legs, and second ventral setae (e).The results of our analysis supports Amrine's (personal communication) decision to describe the species found in Indiana and West Virginia in the USA as Eriophyes n.sp.Mites from Serbia were also compared with some disposed characters of a Bulgarian population (Natchev, 1978).The E. mali from Bulgaria were: 201 μm long, 51 μm wide, had four rayed empodium, genital coverflap with 12 striae and opisthosoma with 78 annuli.The comparison made in this study shows differences in the sizes of certain structures between the European and North American specimens.Namely, E. mali mites from Europe (Bulgaria [Austria?]and Serbia) differ from those of USA and New Zealand origin.

Symptoms and bionomy
E. mali overwinters at the base of buds or under bud scales.Females are the most numerous ovewintering mites (95-98%).Overwintering nymphs are very rare, while eggs and males were not found in buds.About 1000 individuals could be found in a single bud.Mites start to disperse from buds from 8-18 May when average temperatures reach 11-17 °C.Females feed for two weeks on leaf surface and cause blister galls.Blisters can be found not only on leaves, but also on fruits.Females start laying eggs at the beginning of June, and larvae and nymphs appear (emerge) ten days later.Adults of summer generations emerge at the beginning of July.Males are less numerous in comparison with females, 2-5 males : 100 females.There are two generations per season.Second generation is more numerous and around 30 individuals can be found in one gall, while there are 18 individuals per gall in the first generation.One generation develops within 25-30 days.Females are present during the second summer generation (protogyne) and winter (deutogyne).Females start to search for overwintering places at the end of July, but most of them go to overwinter in the middle and at the end of August (Krasinskaya, 1960).Mite feeding creates blister gall formations on leaves (Figure 2).In the centre of each gall there is a hollow which mites enter and go out from (Figure 3).Later, mites feed and develop inside the gall (Figure 4).The percentages of infested leaf surface ranged from 1.6% to 87.6%, the average being 22.4%.Infestation of most of the measured leaves was low with the average rate of 5%, while infestation exceeding 80% was only measured on two leaves (Figure 5).

Figure 3 .
Figure 3. Blisters on apple leaf with openings at the centre (orig.)

Table 2 .
Comparison of characters of Eriophyes mali from different localities(Nalepa 1926, USA, New Zealand and Serbia)