Sample collection

During 2013 and 2014, a directed sampling procedure was carried out in a forest nursery located in Chilpancingo, Guerrero, Mexico, where 20 Pinus patula and P. pseudostrobus seedlings with symptoms of root rot and wilting.

Fungi isolation, purification and preservation

In order to isolate the causative agent, fragments of P. patula and P. pseudostrobus roots with symptoms of dry rot were decontaminated by immersion in a sodium hypochlorite solution at 2 % during 3 minutes; the fragments were washed twice in sterilized distilled water and placed in Petri dishes with a 90 mm diameter in 20 mL of potato-dextrose-agar (PDA) culture medium (Difco) with streptomycin (Sigma^TM). The dishes were placed in incubation at a temperature of 25 °C, under a regime of 12 h light/12 h darkness.

After three days of incubation, the fungal colonies which occurred most frequently were transferred to Petri dishes with a fresh PDA culture medium and placed in incubation under the conditions described above. Once the colonies produced spores, the isolates were purified using the monospore culture technique, transferring a simple spore to a fresh PDA culture medium. Fungal isolates were preserved through the transference of mycelial disks (of 5 mm diameter) to 2 mL cryogenic tubes with 1.5 mL glycerin at 20 % (v/v) and stored at a temperature of -80 °C.

Morphological characterization

The fungal isolates most frequently separated were identified through the morfological characterization of the asexual reproduction structures. For this purpose, they were transferred to a PDA culture medium and incubated at 25 °C under continuous light. After 12 days of inclubation, semipermanent preparations were made in glycerin at 100 % in order to examine their components under the Olympus BX41 compound microscope. 100 macroconidia and 100 microconidia were observed; their shape, size, color and number of septa were recorded. For the formation of phyalides, the fungal isolates had to be cultivated in a potato-agar culture medium for four days. Subsequently, a humid chamber was prepared in a 90 mm Petri dish with blotting paper moistened to saturation point with sterilized distilled water; 5 mm PDA disks with mycelium growth were transferred and incubated during three days, after which the phyalides produced in the humid chamber were observed under the described compound microscope. The fungi were morphologically identified at genus level using specialized codes (^{Barnett and Hunter,1998}); their identification at species level was based on the descriptions by ^{Leslie and Summerell (2006)}.

Pathogenicity test

The pathogenicity of six isolates morphologically identified as Fusarium was determined through the inoculation of 70 one-year-old Pinus patula and P. pseudostrobus seedlings. The inoculation was carried out by immersing the roots in a suspension of conidia (1 x 10⁶ spores mL^-1) during 10 hours. Ten seedlings of each species, whose roots were placed in sterilized distilled water during 10 h, served as controls. After this step was concluded, the seedlings were individually planted in flowerpots with peat moss and agrolite (2:1) and placed in a nursery at a temperature of 20 to 25 °C and a relative humidity of 50-80 %. The pathogenicity test was carried out twice.

The incidence of the disease was determined by the recorded symptoms and the number of dead trees. With these data, the area under the progress curve of the disease (AUDPC) using the trapezoidal integration method (^{Campbell and Madden, 1990}). The severity of the disease was estimated for each fungal isolate and in each of the two Pinus species using the formula of Townsend and Hueberger:

Where:

n = Degree of infection according to the scale

v = Number of plants per category

N = Maximum degree of infection

V = Total number of plants

According to the various degrees of infection, an apparent severity rating scale was devised as follows: Class 0 = 0 % damage (asymptomatic plant); Class 1 = 20 % damage (fallen needles); Class 2 = 40 % damage (chlorosis or yellowing); Class 3 = 60 % damage (dry tips); Class 4 = 20 % damage (wilting); Class 5 = 20 % damage (dead plant). With the results obtained based on the interactions of each of the isolates and the two assessed pine species, disease progress curves were built and adjusted to epidemiological models (Logistic, Exponential, Monomolecular and Gompertz) in order to determine the parameters of intensity at the onset of the disease (0), and the increase rate (r) and thus, to obtain a signal of the form and development structure of the disease (^{Campbell and Madden, 1990}). The epidemiological models and the AUDPC were adjusted using the SAS 9.3 software (^{SAS, 2011}).

DNA extraction, amplification by PCR and sequentiation

Genomic DNA of six fungal isolates assessed in the pathogenicity test and morphologically identified as belonging to the Fusarium genus was extracted by macerating 50 to 100 mg of mycelium from 10 day old colonies. Subsequently, the protocol indicated in the Plant DNeasy Mini Kit (Qiagen^®) extraction kit was followed. The quality of the DNA was verified by electrophoresis in agarose gel at 0.8 % with 0.5 X TBE buffer using 5μL of the DNA and run with 90 volts. The gel was analyzed in a Gel-Docmod 2000 (BIORAD^®) transilluminator.

The universal initiators ITS5/ITS4 were used for the PCR (^{White et al., 1990}). The reaction mixture was prepared at a final volume of 25 μL, with a 1X PCR buffer, 2.5 mM MgCl2, 0.2 mMdNTP, 0.4 μM of each primer, 1U of DNA polymerase (BioTecMol^®) and 100 ng of DNA. The PCR was carried out in a C1000 (BIORAD^®) thermocycler with an initial denaturalization of 2 min at 94 °C, followed by 35 cycles of 30 s at 94 °C, 30 s at 55 °C, 1 min at 72 °C, and a final extension of 10 min at 72°C. The amplified products were verified by electrophoresis in agarose gel at 0.8 %. The gel was analyzed in a Gel-Docmod 2000 (BIORAD^®) transilluminator.

The fragments amplified with the ITS5/ITS4 initiators were purified using the DNA clean and concentrator protocol (Zymo Research^®). For this purpose, 5 volumes of the DNA Binding buffer were added in a 1.5 mL microcentrifuge tube and mixed by inversion. The mixture was transferred to a Zymo-Spin column in a 2 mL colection tube. It was centrifuged for 30 s at 8 000 rpm, and the supernatant was discarded. 200 μL of the Wash DNA buffer were added to the column and centrifuged for 30 s at 8 000 rpm. The supernatant was discarded, and the mixture was transferred to a new 2 mL tube. 60 μL of Elution DNA buffer were added directly to the column, and the product was incubated during 1 min. The column was transferred to a new 1.5 mL tube and the DNA was diluted. The purified DNA fragments were sent to the Corean enterprise Macrogen^® for sequencing. The sequences thus obtained were compared in the database at the NCBI using the BLASTn tool.

Morphological identification

Six isolates were obtained from the symptomatic vegetal material; of these, two types were found to be the most frequently occurring: the first showed white-yellow mycelium growth, and the second, initially white mycelium growth that, however, turned purple-red with time; both growths were velvety. These types of colonies have been recorded for Fusarium species (^{Nelson et al., 1983}; ^{Leslie and Summerell, 2006}).

The isolate with purple-violet mycelium growth in a PDA culture medium (Figure 1A-B) had 19-43 x 3.1-5.4 μm sickle-shaped, hyaline, thin-walled macroconidia with 3-5 septa and with a foot- shaped basal cell (Figure 1C). Elliptic to oval unicellular microconidia (Figure 1D). Short, sharp unbranched monophyalides (Figure 1E). Intercalary and terminal chlamydiospores in pairs (Figure 1F). All the characteristics agreed with those recorded by ^{Leslie and Summerell (2006)} for Fusarium oxysporum.

A) Eight-day colony; B) Reverse of the colony with a purple-red color; C) Macroconidia; D) Microconidia; E) Phialides; F) Chlamydospores

Figure 1 Morphology of colonies and asexual reproductive structures of Fusarium oxysporum Schltdl. taken from Pinus spp. with root rotting symptoms. 

On the other hand, the fungal isolate with white-yellow cottony mycelium growth (Figure 2A-B) exhibited robust hyaline, fusiform macroconidia with 5-6 septa and with a slightly curved and pointed apical cell (Figure 2C). The microconidia were hyaline, oval and kidney-shaped, with 1-2 septa (Figure 2D), and produced in long monophyalides (Figure 2E). The chlamydospores were quickly formed; they were oval, intercalary between hyphae or terminals in the side branches of the hyphae, and occurred individually and in pairs.

All the characteristics agreed with the findings of ^{Leslie and Summerell (2006)} for the Fusarium solani (Mart.) Sacc. species (Teleomorfo: Haematonectria haematococca).

A) Eight-day colony; B) Reverse of the colony with a yellowish color; C) Macroconidia; D) Microconidia; E) Phialides.

Figure 2 Morphology of colonies and asexual reproductive structures of Fusarium solani (Mart.) Sacc. taken from Pinus spp. with root rotting symptoms. 

Analysis of the sequences of ITS regions

The sequences obtained from the DNA of the six fungal isolates morphologically identified as Fusarium were compared with the sequences submitted to the database of the GenBank, and five of them (KU056813, KU056814, KU056816, KU056817, KU056818) showed 99 % identity with F. oxysporum, while only one (KU056815) exhibited 99 % with those of Fusarium solani. This confirmed the results of the morphological characterization, which indicates that both species of the genus of interest are causative agents of root diseases in Pinus spp. in Mexico.

Fusarium oxysporum has been recognized as an infectious agent of Pinus strobus L. in the United States of America (^{Ocamb and Juzwik, 1995}; ^{Ocamb et al., 2002}); P. elliottii Engelm. and P. taeda L. in Argentina (^{Lori and Salerno, 2003}); P. wallichiana A. B. Jacks in India (^{Hassan et al., 2011}); P. halepensis Miller in Algeria (^{Lazreg et al., 2014}), and P. tecunumanii F. Schwerdtf. ex Eguiluz & J. P. Perry in Colombia (^{Herron et al., 2015}). Fusarium solani has also been associated with Pinus strobus tissues in the United States of America (^{Ocamb and Juzwik, 1995}); P. radiata D. Don in New Zealand (^{Dick and Dobbie, 2002}); P. elliottii and P. taeda in Argentina (^{Lori and Salerno, 2003}); P. tropicalis Morelet in Cuba (^{Guerra et al., 2004}), and P. halepensis in Algeria (^{Lazreg et al., 2014}).

Pathogenicity test

Fifteen days after inoculation (DAI), all the plants inoculated with the spores’ suspension exhibited symptoms of wilting, while the control seedlings remained asymptomatic. Fusarium colonies were re-isolated from the inoculated specimens and exhibited the same morphological characteristics as the originally inoculated colonies, whereby Koch’s postulates were completed. This confirmed that Fusarium oxysporum and F. solani are the species of phytopathogenic fungi that cause the symptoms of root rot and wilting of P. patula and P. pseudostrobus seedlings in Guerrero, Mexico. Similarly, ^{Herron et al. (2015)} carried out successful pathogenicity tests with F. circinatum Nirenberg & O’Donnell, F. marasasianum Herron, Marinc. & M. J. Wingf., F. parvisorum Herron, Marinc. & M. J. Wingf. and F. sororula Herron, Marinc. & M. J. Wingf. isolates in Pinus patula seedlings in Colombia, while ^{Latiffah et al. (2009)} identified F. solani and F. oxysporum as the prevailing species in the soils of a forest area in Malasya, but did not determine the pathogenicity of the isolates.

Incidence a severity of the disease

In regard to the incidence of the disease, F. oxysporum isolate 1 had 100 % only in P. pseudostrobus 8 DAI, while F. oxysporum isolate 2 registered the same percentage during the same period for the two Pinus species. Likewise, F. solani had 100 % in both Pinus species 14 DAI (Table 1). This indicates that the F. oxysporum isolate 2 causes disease faster than the other isolates, due to a positive combination of initial intensity of the disease and an increase rate, while this was not the case with the other isolates, in which total infestation occurred less rapidly.

Table 1 Area under the progress curve of the disease (AUDPC) in Pinus patula Schiede ex Schltdl. & Cham. and Pinus pseudostrobus Lindl. seedlings inoculated with five Fusarium oxysporum Schltdl. isolates and a Fusarium solani (Mart.) Sacc. isolate. 

When the severity of each Fusarium isolate was determined, F. oxysporum isolate 1 in P. pseudostrobus had the highest value, while the F. oxysporum isolate 2 was the most virulent in P. patula, having killed the trees 21 DAI. Furthermore, it should be emphasized that the F. solani isolate caused the same effect on the inoculated seedlings of both species. This agrees with the findings of ^{Lazreg et al. (2014)}, who identified seven Fusarium species associated to P. halepensis seedlings, defined significant differences in shoot and root length and in the vigor index in seedlings inoculated with the various isolates; likewise, they pointed out that F. solani stood out from among the other species of the genus for the rapidity with which they colonized the complete roots of the inoculated pine trees.

The logistic model (y = 1 / (1 + ((1-I) / I) * exp (-rL * t)) was the one that best described the progression of symptoms in both pine species in the presence of the Fusarium isolates evaluated over time. In the case of isolate 2 of F. oxysporum in P. pseudostrobus, it was observed that the initial intensity of the disease was higher with respect to P. patula, but the speed of increase of severity was lower. For the F. solani isolate, the two species of pine showed an initial intensity of disease 0.030 % and a rate of increase of severity percentage of 0.32 per day. Therefore, the Fusarium oxysporum isolate 2 was the most virulent, as it grew 0.397 % per day compared to the F. solani isolate, which showed a growth of 0.32 % per day (Table 2). This is indicative that a higher initial intensity disease affects its development; in ABCPE, the initial intensity is related to the amount of inoculum at time 0, but this inoculum will increase as the rate of increase does, which is also subject to the virulence of the pathogen and to the pine species.

Cuadro 2 Ajuste de modelo logístico del progreso de la severidad de cinco aislados de Fusarium oxysporum Schltdl. y un aislado de Fusarium solani (Mart.) Sacc. inoculados en plántulas de Pinus patula Schiede ex Schltdl. & Cham. y Pinus pseudostrobus Lindl. 

Similarly, ^{Herron et al. (2015)} determined that various Fusarium marasasianum, F. parvisorum and F. sororula isolates caused disease in Pinus patula seedlings; furthermore, they pointed out that there was a variation in the pathogenicity and virulence between fungal isolates of the same species.