Collection and handling of diseased material

P. greggi’s diseased material was collected in the Army’s Forest Nursery at Atlangatepec, Tlaxcala (19° 32’ 27.6” N, 98° 10’ 48” W, at an altitude of 2510 m). One centimeter long root bits were collected and washed with sterile distilled water, sodium hypochlorite at 2 %, for 3 min, and thrice with sterile distilled water. The material was dryed with sterile filter paper and was sown, as indicated by ^{Martínez-Álvarez et al. (2012)}, in a potato dextrose agar with antibotics (PDA+A) (0.05 g L^-1 streptomycin sulphate to prevent the development of bacteria).

Morphological identification

Phylogenetic analysis

The sequences of both DNA strands were assembled and edited with BioEdit v. 7.0.5 (^{Hall, 1999}), thus creating a consensus sequence. This sequence was compiled in a Fasta file, using the Profile mode of Clustal W 1.8.1 (^{Thompson et al., 1994}) of the Mega 5.1 software (^{Tamura et al., 2011}), and it was analysed with the method of maximum parsimony (Table 1). Trust values for blocks within the resulting tree were obtained with a bootstrap analysis with 1000 repetitions (^{Felsenstein, 1985}).

Table 1 Species and sequences from the GenBank used to develop the phylogenetic tree. 

^† Han, K. S., S. C. Lee, J. S. Lee, J. W. Soh, and M. J. Park. 2013. Analysis of taxonomic relationships of Fusarium oxysporum f. sp. Based on TEF gene región. Horticultural and Herbal Science. Unpublished. Siti J., and H. Nagao. 2013. Determination of race of Fusarium solani isolated from canker symptom on pumpkin and relationship among isolates from different origins. School of Biogical Sciences University Sains Malaysia. Unpublished. Nayaka, S. C., E. G. Wulff, U. A. C. Shankar, C. N. Mortensen S. R. Niranjana, and H. S. Prakash. 2008. Applied Botany and Biotechnology University of Mysore. India. Unpublished.

Experimental design and statistical analysis

The experiment had a completely randomized design, with a 3 (substrates)X2 (with and without Fusarium) factorial arrangement, six treatments, and four repetitions (Table 2). A container rack was used for each repetition and the experimental unit was made of 20 plants from the center (80 seedlings per treatment). The results were analyzed with the Mixed procedure of SAS, version 9.0 (^{SAS Institute, 2002}) and the means were compared using the Duncan test (p≤0.05).

Table 2 Treatments used in the production of Pinus greggii Engelm., in 170-mL individual container racks. 

Variables evaluated

Germination (emergence) was evaluated over the course of four weeks in February and March 2015, in order to evaluate the effects of pre-emergence treatments. The percentage of germinated seeds was transformed using the arcsine function for analysis. The weekly record of diseased seedlings was used to evaluate post-emergence and the cumulative percentage with drying disease symptoms caused by Fusarium was quantified over the course of 10 weeks, during March, April, and May. Finally, diseased seedlings were transferred to a PDA+A culture medium to verify Koch’s postulates and obtain again the inoculated fungus strain.

Morphological and Molecular Identification

The fungus from the Atlangatepec nursery was identified as Fusarium circinatum Nirenberg & O’Donnell and as a member of the Gibberella fujikuroi (CGF) complex. This pathogen only infects Pinus spp. trees and it is responsible for pitch canker disease in plantations. ^{Wingfield et al. (2008)} and ^{Gordon et al. (2015)} reported it as a significant phytosanitary problem and a threat to nurseries and plantations all over the world.

The sample evaluated showed cottony, white mycelium with an intense violet pigmentation in PDA (Figure 1A and B), 8X3.5 µm oval microconidia without septa (Figure 1H), microconidia aggregated in false heads (Figure 1E), coming out of monophyalides and polyphyalides (Figure 1D and F). Fursarium circinatum can distinguished from F. pseudocircinatum based on the formation of microconidia with short chains instead of false heads, as Nirenberg and O’Donnell (1998) pointed out. The species detected in SNA developed circinas, which are partitioned and curved sterile hypha (Figure 1I). Other species that form these structures are F. pseudocircinatum and F. sterilihyphosum. Fusarium circinatum does not show chlamydospores, but some strains sometimes produce swollen hypha that may look like chlamydospores or pseudochlamydospores (Nirenberg and O’Donnell, 1998; ^{Leslie and Summerell, 2006}). ^{Nelson et al. (1983)} and ^{Pfenning et al. (2014)} report pale orange sporodochium in water agar with carnation leaves; macroconidia, usually from three septa, measure 35X4 µm (Figure 1C and G); there are curved apical cells and underdeveloped basal cells.

Figure 1 Characteristics of Fusarium circinatum. A) White to violet mycelium. B) Violet pigment in the culture medium. C and G) Alantoid macroconidia. D and F) Microconidia’s monophyliades and polyphyliades. E) Microconidia with false heads. H.) Oval microconidia. I) Circinas in SNA medium. 

The molecular identification of the inoculated strain was deposited in the GenBank (accession number MF075250), was aligned with the F. circinatum clade that was isolated from the P. greggii species (Figure 2). Other pine hosts affected by this fungus and molecularly identified as specific initiators were reported in P. taeda with molecular markers MAT1-1 and MAT1-2 (^{Pfenning et al., 2014}), P. montezumae and P. patula with β-tubulin, histone H3, and others (^{Kvas et al., 2009}), and P. radiata with CIRC1A and CIRC4A (^{Martínez-Álvarez et al., 2012}).

Figure 2 Phylogenetic tree of the Fusarium circinatum isolate, with PCR-TEF amplification and its sequences grouped using the MEGA 5.1 software and developed using the method of Maximum Parsimony. 

Effect of Fusarium circinatum in seeds (pre-emergence)

Fusarium circinatum reduced the germination of P. greggii seeds by 43 % (p≤0.01). Therefore, the average germination percentage diminished from 83 (absence of the pathogen) to 48 (after seeds where soaked in F. circinatum). The lower germination (43 %) of the treatments inoculated with F. circinatum was found in substrate S2, while 56 % of the seeds in S3 germinated. ^{Swett and Gordon (2015)} reported that F. circinatum, when it has been isolated from the pine, is able to colonize maize seeds (Zea mays) and observed that up to 98 % of those seeds was infected. The treatements in our study that were not inoculated with F. circinatum show over 80 % germination, without significant differences (p˃0.05) between substrates (Table 3).

Table 3 Germination of Pinus greggii seeds in three substrates. 

Mean values with different letters in the same column are statistically differents, based on the Duncan test (p≤0.05). S1: peat moss, perlite, and vermiculite; S2: sawdust, bark, and peat moss; and, S3: bark, sawdust, and peat moss; the ratio for the three was 60:20:20. DS: standard deviation.

This result matched the reports that F. circinatum reduces the germination of P. radiata (^{Martínez-Álvarez et al., 2012}). ^{Peterson (2008)} indicated that Fusarium arrives at the forest nurseries where conifers are produced, mainly in the seed cover and inside the seed (^{Barrows-Broaddus and Dwinell, 1985}; ^{Storer et al., 1998}) and that Fusarium invades the embryo and kills it (^{Pfenning et al., 2014}).

In the presence of F. circinatum, S1 and S2 seeds had a relatively lower germination percentage than S3. This matched the report made by ^{Hoitink et al. (1997)} that a high rate of peat moss increases the possibility of diseases and root rot.

Effect of F. circinatum in P. greggii seedlings (post-emergence)

The disease became evident 5 weeks after F. circinatum was inoculated during post-emergence (Figure 3A). The symptomatology matched the one observed in the Army’s forest nursery at Atlangatepec (Figure 3B). During the “matchstick” stage, strangulation could be seen in the plant’s apex, which looked dehydrated; after a few days, the seedling could not stand up and died (Figure 3C). ^{Peterson (2008)} and ^{Wingfield et al. (2008)} also observed that conifers are susceptible to this disease and that, during post-emergence, the fungus grows in the vascular tissue and causes the plant to wither and die.

Figure 3 Drying disease caused by Fusarium circinatum. A) Diseased plants with different symptom stages. B) Three-month old seedlings from the nursery at Atlangatepec, Tlaxcala. C) Symptoms during the “matchstick” stage. D). Three-month old diseased plants, after inoculation. E) F. circinatum colonies developed in PDA+A, with inoculated seeds. F) F. circinatum reisolations in one-month old seedlings. 

In 2-3-month-old plants, the main shoot was flaccid and had bent over, the color of the needles had changed from yellow to an intense brownish-grey reddish color, the main root had rotten, and some secondary roots had become brown and their tissue had disintegrated (Figure 3D). These symptoms matched those described by ^{Herron et al. (2015)} and ^{Marín-Cruz et al. (2015)} in seedlings up to their first year and they included withering, fall of the terminal bud, discolouring, and loss of needles.

^{Solano and Brenes (2012)} observed foliage chlorosis or withering of the stem’s apex: these are also symptoms of rotting in the root system (^{Vivas et al., 2009}). The plants also developed white or pale orange sporodochia in the base and along the stem. ^{Peterson (2008)} observed the development of the Fusarium spp. genus with salmon to coraline pink on the base of the stem, root rot during post-emergence, and withered seedlings; this information confirmed the root rot symptoms of P. greggii during the post-emergence stage and in seedlings.

Pathogen recovery

One-hundred percent of the isolations -from 249 non-germinated seeds- were recovered from a PDA medium with antibiotics (Figure 3E). One-hundred fifty-six reisolations were obtained from diseased P. greggii seedlings, out of a total of 169 (93 %) (Figure 3F) of the F. circinatum treatments. This proved the pathogenicity. In a similar study, with F. circinatum in Pinus radiata, a 10 % sample of the experiment’s plants produced 100 % isolations (^{Martínez-Álvarez et al., 2012}). ^{Marín-Cruz et al. (2015)} isolated F. circinatum from P. montezumae and ^{Herron (2015)} isolated it from P. patula, in nursery conditions.

F. circinatum incidence in P. greggii seedlings growing in three substrates

The analysis of the three substrates with F. circinatum inoculum showed that the incidence in P. greggii increased from week 1 to week 10 (Figure 4). Fusarium circinatum had a 22 % incidence in S1 plants, during the first week; in contrast, it had an 8 % and 0 % incidence in S3 and S2 plants, respectively. Incidence in S2 was the lowest during the whole evaluation period and it was not until after the fourth week that it the number of diseased plants reached 25 % (Figure 4).

Figure 4 Weekly incidence of Fusarium circinatum in Pinus greggii plants growing in different substrates. S1: peat moss, perlite, and vermiculite. S2: sawdust, bark, and peat moss; and S3: bark, sawdust, and peat moss; with a 60:20:20 ratio in the three mixtures. 

Since the first week, the percentage of diseased plants was greater in S1 and by the fourth week it had reached 56 % (Figure 4). This shows that the traditional substrate is the most favourable for the development of the disease. In S3, which has a greater rate of pine bark, the percentage of diseased plants was lower during the first six weeks, but then it matched S1.

S2, which was made mainly from sawdust, showed a lower disease incidence. There were no diseased plants during the first week and by the fourth week the incidence only reached 28 %, in contrast with 56 % diseased plants in S1. Although F. circinatum in S2 turned up late, by the tenth week it had equaled S1 and S3 (Figure 4).