Morphological: Morphological identification of the four brown rot fungi is commonly based on morphology and colony characteristics. Identification is possible by combining cultural characteristics, such as growth rate, growth pattern and color, with morphological data, such as conidial dimensions and the length of the germ tube (van Leeuwen and van Kesteren, 1998; De Cal and Melgarejo, 1999).
CPHST Beltsville is close to having a validated molecular method for Monilinia and Monilia spp. When the work instructions are complete, this site will be updated to include a molecular diagnostic method. They are working on two conventional PCR methods that, if done together, can distinguish six Monilinia/Monilia spp. (M. fructicola, M. laxa, M. fructigena, M. polystroma, M. mumecola and M. yunnanensis).
Monilia polystroma could easily be confused with the three brown rot fungi Monilinia species. Monilia polystroma was first classified as Monilinia fructigena. Monilinia laxa (known to occur in the United States) is considered to be more a pathogen of blossoms and twigs than of fruit and primarily occurs on Prunus spp. M. fructigena is mainly a fruit pathogen and primarily occurs on apple, pear, and other pome fruit trees, although it is also found on Prunus spp. M. fructicola (widespread in the United States) is a pathogen of blossoms, twigs, and fruits and mainly affects stone fruits but can occur on apples, pears, and other pome fruits. The color of the pustules on infected plant tissue is buff for M. fructigena and grayish-brown for M. fructicola and M. laxa.
Monilia polystroma is quite similar to Monilinia fructigena but differences do exist (van Leeuwen et al., 2002). Monilia polystroma forms a large number of yellow or buff-colored stromata (van Leeuwen et al., 2002). Monilinia fructigena has the largest macroconidia where the conidia of Monilia polystroma are slightly smaller. Colonies of Monilia polystroma are similar to those of M. fructigena, but black stromatal plates occur on the colonies after incubation for 10-13 days, and Monilia polystroma isolates grow faster than M. fructigena isolates under the same conditions (van Leeuwen et al., 2002).
Other fungi can cause rots with similar symptoms to Monilia polystroma (Penicillium spp., Mucor spp.). Avoid collecting fruits with blue, green, or yellow colored molds or fruit that are "leaking" fluid.
In Progress / Literature-based Diagnostics:
Culture/Isolation: For isolation, the standard procedure is to place pieces of infected material (with or without surface sterilization) on slightly acid agar medium (EPPO, 2009). Isolation of Monilinia spp. from stone fruit and pome fruit surfaces is difficult, however, due to the presence of several fast-growing fungal species. It is also possible to have mixed Monilinia infections. Phillips and Harvey (1975) tested a medium containing pentachloronitrobenzene (PCNB), canned strained peaches, neomycin, streptomycin, agar, and distilled water and found that though it was not totally selective that it could be used to estimate spore density of Monilinia spp. on the surface of fruit. Amiri et al. (2009) developed a new selective medium (acidified potato dextrose agar (PDA) with fosetyl-Al) for recovery and of enumeration of Monilinia spp. from stone fruit.
Molecular: Several molecular methods have been developed to distinguish Monilinia species. Fulton and Brown (1997) and Snyder and Jones (1999) established a PCR-based method of targeting to distinguish M. fructigena from M. fructicola and M. laxa based on the group I intron in the gene for the ribosomal subunit. Subsequent studies, however, showed that these methods were not reliable because some isolates of M. fructicola lack a group I intron in their nuclear rDNA small subunit (Förster and Adaskaveg, 2000; Fulton et al., 1999; Hughes et al., 2000; Cote et al., 2004b).
Other PCR primers and protocols for M. fructicola were published by Förster and Adaskaveg (2000), Boehm et al. (2001), and Ma et al. (2003). However these methods discriminate M. fructicola from M. laxa but have not been validated for distinguishing M. fructicola from M. fructigena. Fluorescent AFLP fingerprinting and inter-simple sequence repeat analysis has been used to examine the genetic diversity of M. fructicola (Fan et al., 2010; Gril et al., 2010).
Ma et al. (2005) developed a pair of PCR primers specific to M. laxa on the basis of the differences in the DNA sequence of the intron 6 of the beta tubulin gene from M. laxa, M. fructicola and other fungal species.
Ioos and Frey (2000) designed species-specific primer pairs for Monilinia fructigena, M. fructicola, and M. laxa based on the ribosomal internal transcribed spacer (ITS) region. Hughes et al. (2000) also developed species-specific primers for Monilinia fructigena, M. fructicola, and M. laxa. An internal control based universal PCR protocol was developed for Monilinia spp., and species-specific primers were designed by using SCAR makers (Gell et al., 2007). Miessner and Stamler (2010) and Hily et al. (2010) developed a primer/primers based on difference in the intron-exon of the cytochrome b gene to distinguish Monilinia fructigena, M. fructicola, and M. laxa. Cote et al. (2004) developed a multiplex PCR that can distinguish Monilinia fructigena, M. fructicola, M. laxa, and Monilia polystroma on inoculated and naturally infected apple and stone fruit. This PCR method uses a common reverse primer (MO 368-5) and three species specific forward primers (MO 368-8R, MO 368-10R, and Laxa - R2) to differentiate the three Monilinia species. In this assay, a 402-bp PCR product for M. fructigena, a 535-bp product for M. fructicola, and a 351-bp product for M. laxa are produced. Furthermore, another specific 425-bp PCR product was amplified, enabling the identification of isolates of Monilia polystroma. Malvarez et al. (2001) were able to use the Cote et al. (2004) primers (prior to their publication) to identify species of Monilinia in Uruguay. Upon comparing the M. fructigena and M. polystroma sequences with the genomic sequence of unknown function previously described by Cote et al. (2004). Petroczy et al. (2012) revealed insertions and substitutions in the M. polystroma sequences. Repetitive sequence motifs were identified, which can be used for differentiation between M. fructigena and M. polystroma.
According to EPPO (2009), the PCR method of Hughes et al. (2000), Ioos and Frey (2000), and Cote et al. (2004) have been shown not to give cross-reaction with Monilia polystroma.
Real-time PCR methods have been developed by Luo et al. (2007) and van Brouwershaven et al. (2010). The Luo et al. (2007) method, which is based on the Ma et al. (2003) primer for M. fructicola, is a SYBR Green assay and has been tested only against M. fructicola, M. laxa, Botrytis cinerea, Botryosphaeria dothidea, and Alternaria alternata. The van Brouwershaven (2010) method is a Taq man assay and has been validated against Monilinia fructigena, M, laxa, M. fructicola, and Monilia polystroma; a FAM-labeled probe will detect M. fructicola while a VIC-labeled probe will detect M. fructigena, M. laxa, and Monilia polystroma as a group. Since the United States currently has both M. fructicola and M. laxa, at present these real-time methods may be of limited utility for the detection of exotic Monilinia or Monilia species.
Seven different PCR methods were tested by Hu et al. (2011) to differentiate Monilinia spp. None of the six molecular tools alone were able to distinguish all five Monilinia species (M. fructigena, M. fructicola, M. laxa, M. yunnaensis, and M. mumecola) (Ioos and Frey 2000; Ma et al. 2003, 2005; Cote et al., 2004; Gell et al., 2007; Miessner and Stammler, 2010; Hily et al., 2010). Note: The authors didn"t test Monilia polystroma.
M. fructigena, M. fructicola, and M. laxa were reliably differentiated by the methods of Ioos and Frey (2000), Miessner and Stammler (2010), and Hily et al. (2010). However, neither of these methods was able to distinguish M. fructigena from M. yannanensis. Likewise, the methods developed by Ioos and Frey (2010), Ma et al. (2003, 2005) did not distinguish between M. mumecola and M. laxa. The method developed by Hily et al. (2010) did not distinguish M. mumecola from M. fructicola. Additionally, the methods of Miessner and Stammler (2010) and Hily et al. (2010) did not distinguish between M. yunnanensis and M. laxa.
Hu et al. recently (2011) developed an additional multiplex PCR to distinguish M. fructicola from M. mumecola, M. yunnanensis in China. Additional work needed to see if these primers distinguish M. fructigena, Monilinia laxa, and Monilia polystroma, because the authors did not find these species in China and did not present any specific data for these species.